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

Abstract

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming ␣-subunit or certain auxiliary ␤-subunits. Upon coexpression, Kv␤1.1 was found to endow non-inactivating members of the Kv1␣ family with fast inactivation via its unique N terminus. Here we investigated structure and functional properties of the Kv␤1.1 N terminus (amino acids 1-62, ␤N-(1-62)) using NMR spectroscopy and patch clamp recordings. ␤N-(1-62) showed all hallmarks of N-type inactivation: it inactivated non-inactivating Kv1.1 channels when applied to the cytoplasmic side as a synthetic peptide, and its interaction with the ␣-subunit was competed with tetraethylammonium and displayed an affinity in the lower micromolar range. In aequous and physiological salt solution, ␤N-(1-62) showed no well defined three-dimensional structure, it rather existed in a fast equilibrium of multiple weakly structured states. These structural and functional properties of ␤N-(1-62) closely resemble those of the "unstructured" ID from Shaker B, but differ markedly from those of the compactly folded ID of the Kv3.4 ␣-subunit.Fast N-type inactivation of voltage-gated potassium (Kv) 1 channels shapes the action potential, governs the firing rate (spiking), and controls signal propagation in central neurons (1). Biophysically, N-type inactivation has long served as the model for gating transitions in ion channels and is realized by a "ball plug-in" mechanism. In this mechanism a protein domain termed "inactivation gate" or "inactivation ball" binds to its receptor at the inner vestibule of the open channel and thereby occludes the ion pathway (2-5). Such inactivation gates have been localized in the N terminus of various Kv␣ subunits and were shown to be functional entities, i.e. they conferred rapid inactivation to "ball-less" Kv␣ subunits when applied to the cytoplasmic side of the channels as synthetic peptides (5-8). Identical to protein-harbored inactivation domains, the synthetic gates interacted with channels in the open state, blocked the pore with low voltage dependence, and were competed with the channel blocker tetraethylammonium (TEA) (9 -11). Recently, the structures of the inactivation domains (ID) from Kv1.4 and Kv3.4 were determined with NMR spectroscopy. Both IDs were found to exhibit well defined and compact folding in aequous solution (12). In contrast, the ID from Shaker B showed no unique, folded structure (13,14).Besides with Kv␣ subunits owning an N-terminal ID, fast inactivation was observed for a subset of non-inactivating Kv␣1 channels when coexpressed with certain ␤-subunits (15-17). These auxiliary subunits constitute a family of cytoplasmic proteins (subdivided into subfamilies Kv␤1, -2, and -3) that are made up of two distinct regions: a highly conserved core region that shows homolo...

NMR Structure of the “Ball-and-chain” Domain of KCNMB2, the β2-Subunit of Large Conductance Ca2+- and Voltage-activated Potassium Channels

“…These correlations between structural and functional properties are consistent with our earlier observations that well ordered IDs exhibit a faster k on and a much slower k off than unfolded domains (17,19). The latter seems to be caused either by the higher number of molecular contacts (hydrogen bonds, etc.)…”

“…1B, the structure of BK␤ 2 N is accompanied by functional properties that are unique with respect to those of Kv-derived IDs. Thus, the association rate of BK␤ 2 N with its receptor on the channel is very similar to that observed with the unstructured IDs of Kv␤1.1 or Shaker B, but considerably slower than that determined for the compactly folded Kv3.4-ID (17,19,33). The dissociation rate of BK␤ 2 N, on the other hand, is more than 10-fold lower than that of Kv␤1.1-ID or the ID of Shaker B and even about 2-fold lower than that of Kv3.4-ID.…”

“…1D and Ref. 19). Next we investigated the structural properties of BK␤ 2 N in solution with NMR spectroscopy.…”

A Marriage of Convenience: β-Subunits and Voltage-dependent K+ Channels

“…The structure of the isolated Kv␤1 N terminus (amino acids 1-62) was solved using NMR spectroscopy (17). The N terminus of Kv␤1.1 does not exhibit a well defined, unique, threedimensional structure, indicating a fast conformational equilibrium between weakly structured substrates.…”

Solution Structure and Function of the “Tandem Inactivation Domain” of the Neuronal A-type Potassium Channel Kv1.4

“…This classical gating process is brought about by a "plugin" mechanism in which a protein domain termed "inactivation ball" occludes the ion pathway through interaction with a receptor site in the pore of the channel (6). Structure-function analysis showed: (i) that inactivation domains (ID) are formed by the first 20 -40 amino acids at the N terminus of certain Kv␣ (7)(8)(9)(10) and Kv␤ subunits (11,12); (ii) that IDs inactivate channels independent of whether they are tethered to the protein or offered to the channel as a synthetic peptide (8,9,13,14); (iii) that IDs enter the channel pore and compete with open pore blockers such as tetraethylammonium (TEA) (11,(15)(16)(17); and finally (iv) that NMR-derived solution structures of the known IDs vary from a well ordered and compact folding as in Kv3.4 ID (18) to the disordered and highly flexible ID from ShakerB (19). Different from other ␣/␤ subunits that harbor one ID in their N terminus, Kv1.4 was found to be equipped with two IDs with the ability to endow the channel with rapid inactivation (Fig.…”