Voltage-dependent Gating Rearrangements in the Intracellular T1–T1 Interface of a K+ Channel

Abstract

The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a “hanging gondola” below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1–S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn2+ site thiolate groups in the T1–T1 interface; therefore, we concluded that the T1–T1 interface is functionally active and not protected by Zn2+ (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55–69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (k MTSET) is dramatically enhanced in the activated state relative to the resting and inactivated states (∼260- and ∼47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, k MTSET is steeply voltage dependent in a manner that is precisely correlated with the peak conductance–voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn2+ site. Furthermore, cross-linking of cysteine pairs across the T1–T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1–T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function.

Three-dimensional structure of the KChIP1–Kv4.3 T1 complex reveals a cross-shaped octamer

“…Kv4 inactivation is thought to arise from the concerted action of the N-and C-terminal cytoplasmic domains 36 . Although the Kv4 N terminus can influence inactivation in the absence of KChIPs 37,38 , the intimate association of T1N and KChIP excludes the possibility that KChIP-Kv4 inactivation occurs by a balland-chain mechanism and supports the idea that T1 conformational changes are coupled to Kv channel gating 35,39,40 . Exactly how KChIPs, T1 and the α subunit's transmembrane segments are mechanistically coupled remains to be explained.…”

The dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting β-subunit of neuronal KV4.2 channels

“…[7][8][9] The membrane-embedded VSD and PD are essential for voltagedependent gating, 10 whereas the cytoplasmic T1 domain plays multiple regulatory and structural roles. [11][12][13][14] Diverse auxiliary β-subunits may interact with these domains specifically to modify the K V channel phenotype and dictate its physiological role in excitable tissues. 4,[15][16][17][18][19][20] Neuronal K V 4 channels underlie the somatodendritic A-type K + current (I SA ).…”

Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

“…Similar effects were found when studying mutations in T1 in Kv1-type channels [231][232][233]. The role T1 plays for gating is not clear but these studies imply that channel rearrangement during gating is not restricted to the transmembrane domains but also involves T1 [234]. Considering the effects from intracellular C-terminal Mg 2+ binding, one could speculate that the T1 domain may also interact electrostatically with the VSD and that this interaction could be influenced by charge-altering substitutions.…”