Inactivation of a Voltagedependent K+ Channel by β Subunit

Abstract

Kv1.1/Kv␤1.1 (␣␤) K ؉ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the ␣ subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of ␣, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of ␣. Truncation of the last six amino acids resulted in ␣␤ channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the ␤ subunit.In contrast with the well understood structural determinants within the core of voltage-gated K ϩ (Kv) channel proteins that underlie many functional properties (e.g. voltage-sensing regions, pore region; Refs. 1-5), much less is known about the role of extrinsic factors in determining Kv channel function. One such factor is the interaction of ion channels with the cytoskeleton, which could affect structural and functional organization of neuronal elements. Recently, a family of membrane-associated putative guanylate kinases were shown to bind directly to K ϩ channels of the Shaker (Kv1) family and to induce their clustering in COS7 cells (6,7). This family includes PSD-95, an abundant synaptic protein found both preand postsynaptically, and SAP97, a protein found in axons of neurons as well as in non-neuronal cells (for reviews, see Refs. 8 and 9). These data together with the demonstration of colocalization of the channels with PSD-95 (6, 10) in the brain, particularly in nerve terminal plexuses of basket cells in the cerebellum, provide correlative evidence of a direct association in vivo. Apart from a probable role in clustering, it is not known whether the association with PSD-95 has a functional role of modulating the biophysical characteristics of the channel.For several years now our interest has been the modulation by direct phosphorylation of the voltage-gated K ϩ channel Kv1.1 (originally cloned from rodent brain cD...

NMR Structure and Functional Characteristics of the Hydrophilic N Terminus of the Potassium Channel β-Subunit Kvβ1.1

“…␤N-(1-62) induced almost complete inactivation, whereas inactivation mediated by the entire Kv␤1.1 protein was found to be considerably less effective (15,16,35,36). This discrepancy might reflect some variation in binding of Kv␤1.1 to the N terminus of Kv1.1 or might be because of interaction of the ␣-␤ complex with other proteins affecting the mobility of the ␤-inactivation domain.…”

Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvβ2-subunit

“…Coexpression of Kv␤ with Kv␣ enhances channel synthesis and cell surface targeting (21)(22)(23). Coexpression of either Kv␤1 or -3 with Kv␣ results in a dramatic acceleration of Kv channels inactivation (24,25). The N termini of Kv␤1 and Kv␤3, which are highly homologous to the Kv inactivation peptide endogenous to Kv␣, are responsible for this enhanced inactivation.…”

Activation of a Metabotropic Glutamate Receptor and Protein Kinase C Reduce the Extent of Inactivation of the K+Channel Kv1.1/Kvβ1.1 via Dephosphorylation of Kv1.1

“…This effect is opposite that shown by us for a constitutively active kinase (yet unidentified) or for a stimulated PKA that causes an increase in the extent of inactivation by phosphorylating Ser-446 that impairs interaction between the channel and the microfilaments (8). Part of the interaction with the microfilaments was shown to be mediated via a post-synaptic density-95-like protein that interacts with the C-terminal end of Kv1.1 (9). A similar phenomenon was described in mammalian cells 2) or from uninjected oocytes (lane 4).…”

“…44). Notably, a post-synaptic density-95-like endogenous protein was shown by us to interact with the channel and to affect its extent of inactivation (9). The effect of glutamate on the current amplitudes was small compared with that of PMA, possibly because it was somewhat occluded by the effect exerted by spontaneous coupling of the receptor without glutamate to the signaling machinery, since coexpression of the receptor with the channel reduced the current amplitudes significantly even in the absence of agonist.…”

“…Recently we showed (8) that the extent of fast inactivation of the heteromultimeric Kv1.1/Kv␤1.1 (␣␤) channel expressed in Xenopus oocytes is regulated by the basal and PKAinduced phosphorylations of the ␣ subunits that affect the interaction of the channel with microfilaments. Part of the interaction is probably mediated by a native post-synaptic density-95-like protein of the oocyte that recognizes the C-terminal end of the ␣ subunit (9).…”