The Kv2.1 K+ channel targets to the axon initial segment of hippocampal and cortical neurons in culture and in situ

<p>Abstract</p> <p>Background</p> <p>The Kv2.1 delayed-rectifier K⁺channel regulates membrane excitability in hippocampal neurons where it targets to dynamic cell surface clusters on the soma and proximal dendrites. In the past, Kv2.1 has been assumed to be absent from the axon initial segment.</p> <p>Results</p> <p>Transfected and endogenous Kv2.1 is now demonstrated to preferentially accumulate within the axon initial segment (AIS) over other neurite processes; 87% of 14 DIV hippocampal neurons show endogenous channel concentrated at the AIS relative to the soma and proximal dendrites. In contrast to the localization observed in pyramidal cells, GAD positive inhibitory neurons within the hippocampal cultures did not show AIS targeting. Photoactivable-GFP-Kv2.1-containing clusters at the AIS were stable, moving <1 <it>μ</it>m/hr with no channel turnover. Photobleach studies indicated individual channels within the cluster perimeter were highly mobile (FRAP <it>τ </it>= 10.4 ± 4.8 sec), supporting our model that Kv2.1 clusters are formed by the retention of mobile channels behind a diffusion-limiting perimeter. Demonstrating that the AIS targeting is not a tissue culture artifact, Kv2.1 was found in axon initial segments within both the adult rat hippocampal CA1, CA2, and CA3 layers and cortex.</p> <p>Conclusion</p> <p>In summary, Kv2.1 is associated with the axon initial segment both <it>in vitro </it>and <it>in vivo </it>where it may modulate action potential frequency and back propagation. Since transfected Kv2.1 initially localizes to the AIS before appearing on the soma, it is likely multiple mechanisms regulate Kv2.1 trafficking to the cell surface.</p

Size of Cell-Surface Kv2.1 Domains is Governed by Growth Fluctuations

AbstractThe Kv2.1 voltage-gated potassium channel forms stable clusters on the surface of different mammalian cells. Even though these cell-surface structures have been observed for almost a decade, little is known about the mechanism by which cells maintain them. We measure the distribution of domain sizes to study the kinetics of their growth. Using a Fokker-Planck formalism, we find no evidence for a feedback mechanism present to maintain specific domain radii. Instead, the size of Kv2.1 clusters is consistent with a model where domain size is established by fluctuations in the trafficking machinery. These results are further validated using likelihood and Akaike weights to select the best model for the kinetics of domain growth consistent with our experimental data

Functional consequences of the variable stoichiometry of the Kv1.3-KCNE4 complex

The voltage-gated potassium channel Kv1.3 plays a crucial role during the immune response. The channel forms oligomeric complexes by associating with several modulatory subunits. KCNE4, one of the five members of the KCNE family, binds to Kv1.3, altering channel activity and membrane expression. The association of KCNEs with Kv channels is the subject of numerous studies, and the stoichiometry of such associations has led to an ongoing debate. The number of KCNE4 subunits that can interact and modulate Kv1.3 is unknown. KCNE4 transfers important elements to the Kv1.3 channelosome that negatively regulate channel function, thereby fine-tuning leukocyte physiology. The aim of this study was to determine the stoichiometry of the functional Kv1.3-KCNE4 complex. We demonstrate that as many as four KCNE4 subunits can bind to the same Kv1.3 channel, indicating a variable Kv1.3-KCNE4 stoichiometry. While increasing the number of KCNE4 subunits steadily slowed the activation of the channel and decreased the abundance of Kv1.3 at the cell surface, the presence of a single KCNE4 peptide was suffcient for the cooperative enhancement of the inactivating function of the channel. This variable architecture, which depends on KCNE4 availability, differentially affects Kv1.3 function. Therefore, our data indicate that the physiological remodeling of KCNE4 triggers functional consequences for Kv1.3, thus affecting cell physiology

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses

The C-terminal domain of Kv1.3 regulates functional interactions with the KCNE4 subunit

The voltage-dependent K+ channel Kv1.3 (also known as KCNA3), which plays crucial roles in leukocytes, physically interacts with KCNE4. This interaction inhibits the K+ currents because the channel is retained within intracellular compartments. Thus, KCNE subunits are regulators of K+ channels in the immune system. Although the canonical interactions of KCNE subunits with Kv7 channels are under intensive investigation, the molecular determinants governing the important Kv1.3- KCNE4 association in the immune system are unknown. Our results suggest that the tertiary structure of the C-terminal domain of Kv1.3 is necessary and sufficient for such an interaction. However, this element is apparently not involved in modulating Kv1.3 gating. Furthermore, the KCNE4-dependent intracellular retention of the channel, which negatively affects the activity of Kv1.3, is mediated by two independent and additive mechanisms. First, KCNE4 masks the YMVIEE signature at the C-terminus of Kv1.3, which is crucial for the surface targeting of the channel. Second, we identify a potent endoplasmic reticulum retention motif in KCNE4 that further limits cell surface expression. Our results define specific molecular determinants that play crucial roles in the physiological function of Kv1.3 in leukocytes