Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

Kv3 (Shaw) channels regulate rapid spiking, transmitter release and dendritic integration of many central neurons. Crucial to functional diversity are the complex targeting patterns of channel proteins. However, the targeting mechanisms are not known. Here we report that the axon-dendrite targeting of Kv3.1 is controlled by a conditional interaction of a C-terminal axonal targeting motif (ATM) with the N-terminal T1 domain and adaptor protein ankyrin G. In cultured hippocampal neurons, although the two splice variants of Kv3.1, Kv3.1a and Kv3.1b, are differentially targeted to the somatodendritic and axonal membrane, respectively, the lysine-rich ATM is surprisingly common for both splice variants. The ATM not only directly binds to the T1 domain in a Zn 2ϩ -dependent manner, but also associates with the ankyrin-repeat domain of ankyrin G. However, the full-length channel proteins of Kv3.1b display stronger association to ankyrin G than those of Kv3.1a, suggesting that the unique splice domain at Kv3.1b C terminus influences ATM binding to T1 and ankyrin G. Because ankyrin G mainly resides at the axon initial segment, we propose that it may function as a barrier for axon-dendrite targeting of Kv3.1 channels. In support of this idea, disrupting ankyrin G function either by over-expressing a dominant-negative mutant or by siRNA knockdown decreases polarized axon-dendrite targeting of both Kv3.1a and Kv3.1b. We conclude that the conditional ATM masked by the T1 domain in Kv3.1a is exposed by the splice domain in Kv3.1b, and is subsequently recognized by ankyrin G to target Kv3.1b into the axon.

Section: Direct Interaction Between the T1 Domain And The Atm Regionsupporting

confidence: 64%

The Axon–Dendrite Targeting of Kv3 (Shaw) Channels Is Determined by a Targeting Motif That Associates with the T1 Domain and Ankyrin G

Xu¹,

Cao²,

Xiao³

et al. 2007

“…Bearing in mind that Syx binds at the CTs (Regev et al, 2009), this suggested a possible cross talk between closely positioned N and C termini. Notably, N-C close associations that are involved in the gating and activation of many channel types have been demonstrated (Varnum and Zagotta, 1997;Schulte et al, 1998;Tucker and Ashcroft, 1999;Qu et al, 2000;Lippiat et al, 2002;Tsuboi et al, 2004;Kobrinsky et al, 2006). Furthermore, using FRET analysis, our laboratory has recently demonstrated that a Kv2.1 regulatory protein modulates channel gating via effects on the association between the termini (Lvov et al, 2009).…”

Section: Discussionmentioning

confidence: 94%

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

Etzioni¹,

Siloni²,

Chikvashvilli³

et al. 2011

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.

“…This suggests that phosphorylation-dependent modulation of Kv2.1 requires N/C interaction. Recent studies using fluorescence resonance energy transfer demonstrated N/C interaction within Kv2.1 and suggested dynamic changes in this interaction during voltage-dependent gating (Kobrinsky et al, 2006). Analyses of the effects of altering phosphorylation state on Kv2.1 N/C interaction using such sensitive techniques may clarify the role of C-terminal phosphorylation in regulating this interaction and channel gating.…”

Section: Discussionmentioning

confidence: 99%

Interdomain Cytoplasmic Interactions Govern the Intracellular Trafficking, Gating, and Modulation of the Kv2.1 Channel

Mohapatra¹,

Siino²,

Trimmer³

2008

Voltage-gated potassium (Kv) channels comprise four transmembrane ␣ subunits, often associated with cytoplasmic ␤ subunits that impact channel expression and function. Here, we show that cell surface expression, voltage-dependent activation gating, and phosphorylation-dependent modulation of Kv2.1 are regulated by cytoplasmic N/C interaction within the ␣ subunit. Kv2.1 surface expression is greatly reduced by C-terminal truncation. Tailless Kv2.1 channels exhibit altered voltage-dependent gating properties and lack the bulk of the phosphorylation-dependent modulation of channel gating. Remarkably, the soluble C terminus of Kv2.1 associates with tailless channels and rescues their expression, function, and phosphorylation-dependent modulation. Soluble N and C termini of Kv2.1 can also interact directly. We also show that the N/C-terminal interaction in Kv2.1 is governed by a 34 aa motif in the juxtamembrane cytoplasmic C terminus, and a 17 aa motif located in the N terminus at a position equivalent to the ␤ subunit binding site in other Kv channels. Deletion of either motif disrupts N/C-terminal interaction and surface expression, function, and phosphorylationdependent modulation of Kv2.1 channels. These findings provide novel insights into intrinsic mechanisms for the regulation of Kv2.1 trafficking, gating, and phosphorylation-dependent modulation through cytoplasmic N/C-terminal interaction, which resembles ␣/␤ subunit interaction in other Kv channels.