Arrangement of Kv1 α subunits dictates sensitivity to tetraethylammonium

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity

Distinctive Role of KV1.1 Subunits in the Biology and Functions of Low Threshold K+ Channels with Implication for Neurological Disease

This document is the Accepted Manuscript version of the following article: Saak V. Ovsepian; Marie LeBerre; Volker Steuber; Valerie B. O’Leary; Christian Leibold; & J. Oliver Dolly; ‘Distinctive role of KV1.1 subunit in the biology and functions of low threshold K+ channels with implications for neurological disease’, Pharmacology & Therapeutics, Vol. 159, March 2016, pp. 93-101. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ The version of record is available on line at doi: http:dx.doi.org/10.1016/j.pharmthera.2016.01.005 © 2016 Elsevier Inc. All rights reserved.The diversity of pore-forming subunits of KV1 channels (KV1.1–KV1.8) affords their physiological versatility and predicts a range of functional impairments resulting from genetic aberrations. Curiously, identified so far human neurological conditions associated with dysfunctions of KV1 channels have been linked exclusively to mutations in the KCNA1 gene encoding for the KV1.1 subunit. The absence of phenotypes related to irregularities in other subunits, including the prevalent KV1.2 subunit of neurons is highly perplexing given that deletion of the corresponding kcna2 gene in mouse models precipitates symptoms reminiscent to those of KV1.1 knockouts. Herein, we critically evaluate the molecular and biophysical characteristics of the KV1.1 protein in comparison with others and discuss their role in the greater penetrance of KCNA1 mutations in humans leading to the neurological signs of episodic ataxia type 1 (EA1). Future research and interpretation of emerging data should afford new insights towards a better understanding of the role of KV1.1 in integrative mechanisms of neurons and synaptic functions under normal and disease conditionsPeer reviewedFinal Accepted Versio

Surface biotinylation of heteromeric Kv1 concatemers expressed in CHO cells

Concatenated constructs of Kv1.1-1.2 or Kv1.1-1.2-1.2-1.2 () were expressed CHO cells by electroporation of RNA transcripts from a Semliki Forest virus vector. After 48 h, electroporated cells were harvested in PBS buffer containing 5 mM EDTA. Cell surface biotinylation was performed with sulfo-NHS-LC-biotin (Pierce Chemical Co.), as recommended by the manufacturer. Membrane proteins solubilized with 2% Triton X-100 were precipitated using streptavidin-agarose (Pierce Chemical Co.). Bound proteins were dissolved in lithium dodecyl sulfate sample buffer (Invitrogen) and SDS-PAGE and Western blotting with anti-Kv1.2 antibody were performed, as outlined elsewhere (). Lanes: 1, biotinylated Kv1.1-1.2; 2, total extract from cells expressing Kv1.1-1.2; 3, biotinylated Kv1.1-1.2-1.2-1.2; and 4, total extract from cells expressing Kv1.1-1.2-1.2-1.2.<p><b>Copyright information:</b></p><p>Taken from "How to Validate a Heteromeric Ion Channel Drug Target: Assessing Proper Expression of Concatenated Subunits"</p><p></p><p>The Journal of General Physiology 2008;131(5):415-420.</p><p>Published online Jan 2008</p><p>PMCID:PMC2346572.</p><p></p

A Defined Heteromeric KV1 Channel Stabilizes the Intrinsic Pacemaking and Regulates the Efferent Code of Deep Cerebellar Nuclear Neurons to Thalamic Targets

The output of the cerebellum to the motor axis of the central nervous system is orchestrated mainly by synaptic inputs and intrinsic pacemaker activity of deep cerebellar nuclear (DCN) projection neurons. Herein, we demonstrate that the soma of these cells is enriched with KV1 channels produced by mandatory multi-merization of KV1.1, 1.2 α and KV β2 subunits. Being constitutively active, the K+ current (IKV1) mediated by these channels stabilizes the rate and regulates the temporal precision of self-sustained firing of these neurons. Placed strategically, IKV1 provides a powerful counter-balance to prolonged depolarizing inputs, attenuates the rebound excitation, and dampens the membrane potential bi-stability. Somatic location with low activation threshold render IKV1 instrumental in voltage-dependent de-coupling of the axon initial segment from the cell body of projection neurons, impeding invasion of backpropagating initial segment action potentials into the somato-dendr itic compartment. The latter also promotes the dominance of clock like somatic pace-making in driving the regenerative firing activity of these neurons, to encode time variant inputs with high fidelity. Through the use of multi-compartmental modeling and retro-axonal labeling, the physiological significance of the described functions for processing and communication of information from the lateral DCN to thalamic relay nuclei is establishedPeer reviewedFinal Accepted Versio

Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3

1. We investigated the action of TsTX-Kα on cloned Kv1.3 channels of the Shaker subfamily of voltage-gated potassium channels, using the voltage–clamp technique. Highly purified TsTX-Kα was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX-Kα blocks Kv1.3 with high affinity in two expression systems. 2. TsTX-Kα blockade of Kv1.3 channels expressed in Xenopus oocytes was found to be completely reversible and to exhibit a pH dependence. The K(D) was 3.9 nM at pH 7.5, 9.5 nM at pH 7.0 and 94.5 nM at pH 6.5. 3. The blocking properties of TsTX-Kα in a mammalian cell line (L929), stably transfected to express Kv1.3, were studied using the patch–clamp technique. In this preparation, the toxin had a K(D) of 19.8 nM at pH 7.4. 4. TsTX-Kα was found to affect neither the voltage-dependence of activation, nor the activation and deactivation time constants. The block appeared to be independent of the transmembrane voltage and the toxin did not interfere with the C-type inactivation process. 5. Taken as a whole, our findings indicate that TsTX-Kα acts as a simple blocker of Kv1.3 channels. It is concluded that this toxin is a useful tool for probing not only the physiological roles of Kv1.2, but also those mediated by Kv1.3 channels