Kv4 channel complexes mediate the neuronal somatodendritic A-type K(+) current (I(SA)), which plays pivotal roles in dendritic signal integration. These complexes are composed of pore-forming voltage-gated alpha-subunits (Shal/Kv4) and at least two classes of auxiliary beta-subunits: KChIPs (K(+)-Channel-Interacting-Proteins) and DPLPs (Dipeptidyl-Peptidase-Like-Proteins). Here, we review our investigations of Kv4 gating mechanisms and functional remodeling by specific auxiliary beta-subunits. Namely, we have concluded that: (1) the Kv4 channel complex employs novel alternative mechanisms of closed-state inactivation; (2) the intracellular Zn(2+) site in the T1 domain undergoes a conformational change tightly coupled to voltage-dependent gating and is targeted by nitrosative modulation; and (3) discrete and specific interactions mediate the effects of KChIPs and DPLPs on activation, inactivation and permeation of Kv4 channels. These studies are shedding new light on the molecular bases of I(SA) function and regulation.

Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe 3؉ no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe 3؉ may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe 3؉ homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

Gating of voltage-dependent K ϩ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn 2 ϩ coordination. Also, added Zn 2 ϩ or a potent Zn 2 ϩ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ‫ف‬ 200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact ␣ -subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1-T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn 2 ϩ binding. I N T R O D U C T I O NActivation of voltage-gated potassium channels (Kv channels) is directly controlled by the movements of their S4 voltage sensors, and a subsequent concerted conformational change that opens an internal gate (Yellen, 1998;Horn, 2000;Bezanilla and Perozo, 2003). The bundle-crossing of four transmembrane S6 segments constitutes the main activation gate that controls K ϩ passage at the internal opening of the tetrameric pore structure (Jiang et al., 2002;Webster et al., 2004). Just beneath the main activation gate, the NH 2 -terminal tetramerization domain (T1) of Kv channels is a fourfold symmetric structure that is responsible for the subfamily-specific coassembly of Kv subunits (Li et al., 1992;Shen et al., 1993). The "side windows" between the T1 domain and the transmembrane core domain provide direct access to the internal mouth of the pore (Kreusch et al., 1998;Gulbis et al., 2000;Kobertz et al., 2000;Sokolova et al., 2001;Kim et al., 2004a). Recent studies have suggested that the T1 domain and other intracellular regions also contribute to the function of Kv channels (Cushman et al., 2000;Gulbis et al., 2000;Minor et al., 2000;Kurata et al., 2002;Hatano et al., 2003;Wray, 2004). However, the underlying molecula...

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.

Thermostability is important for the thermoactivity of proteins including enzymes. However, it is still challenging to pinpoint the specific structural factors for different temperature thresholds to initiate their specific structural and functional perturbations. Here, graph theory was used to investigate how the temperature-dependent noncovalent interactions as identified in the structures of aldolase B and its prevalent A149P mutant could form a systematic fluidic grid-like mesh network with topological grids to regulate the structural thermostability and the functional thermoactivity upon cyclization against decyclization in an extended range of a subunit. The results showed that the biggest grid may determine the melting temperature thresholds for the changes in their secondary and tertiary structures and specific catalytic activities. Further, a highly conserved thermostable grid may serve as an anchor to secure the flexible active site to achieve the specific thermoactivity. Finally, higher grid-based systematic thermal instability may disfavor the thermoactivity. Thus, this computational study may provide critical clues for the structural thermostability and the functional thermoactivity of proteins including enzymes.

No abstract

Curcumin potentiates cystic fibrosis transmembrane conductance regulator (CFTR) activation in an ATP-independent but phosphorylation-dependent manner. The underlying molecular mechanisms are unclear. Here, HEK-293T cells cultured in an Fe(3+)-containing medium were transiently transfected with CFTR constructs, and the role of the inhibitory Fe(3+) bridge between intracellular loop 3 and the regulatory domain of CFTR in this pathway was investigated. The results showed that ethylenediaminetetraacetic acid (EDTA) stimulated phosphorylation-dependent CFTR activation and the stimulation was suppressed by the deletion of the regulatory domain or the insertion of a C832A mutation that removes the Fe(3+)-binding interface. Furthermore, curcumin potentiation of CFTR was significantly weakened not only by Fe(3+)-insensitive mutations at the interface between the regulatory domain and intracellular loop 3 but also by N-ethylmaleimide or EDTA pretreatment that removes Fe(3+). More importantly, potentiation of CFTR was completely suppressed by sufficient Fe(3+). Finally, the insertion of Fe(3+)-insensitive H950R/S768R increased the curcumin-independent activity of ΔF508 but weakened its curcumin potentiation. Thus, Fe(3+) homeostasis in epithelia may play a critical role in regulating CFTR activity, and targeting Fe(3+)-chelating potentiators may direct new therapies for cystic fibrosis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATPdriven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating. The cystic fibrosis transmembrane conductance regulator (CFTR)2 chloride channel is widely distributed in the human organs, including the heart, and mediates the electric response to ATP and protein kinase A or C. As shown in Fig. 1, this protein has two membrane-spanning domains (MSD1 and MSD2), two intracellular nucleotide-binding domains (NBD1 and NBD2), and a unique regulatory (R) domain, although it belongs to the human C subfamily of ATP-binding cassette transporters (1, 2). Each MSD consists of six transmembrane helical segments probably extended to four cytoplasmic loops (3). Although recent studies have strongly suggested structural similarities between CFTR and bacterial transporters Sav1866 and MsbA (3-5), three-dimensional structural information about the whole protein is still unavailable except for the crystal structure of the isolated NBD1 (6). Furthermore, the exact location and relative orientation of the R domain in the whole protein are also unclear because this domain lacks a stably folded globular structure and thus is disordered (7,8).Ion transport of CFTR is triggered by not only ATP binding and hydrolysis at the interface of a NBD1-NBD2 dimer but also phosphorylation by protein kinase A (PKA) (9). Structures of bacterial NBD homodimers indicate two ATP-binding sites at the NBD1-NBD2 interface, and each site is composed of residues from both NBDs (10). However, most PKA phosphorylation sites are mainly found in the R domain (6, 11).CFTR activity is tightly controlled by interdomain interactions. Several thiol-specific cross-linking studies, based on the crystal structures of Sav1866 and MsbA, have shown that the NBD1-NBD2 dimerization drives channel opening (12). However, chemical cross-linking of NBDs to cytoplasmic loops (CLs) inhibits channel activity (Fig. 1) (5, 13, 14). Recent structural studies of CFTR and other ATP-binding cassette transporters suggested rearrangements of CLs that couple dimerization of the NBDs to a change in the MSDs from an inward to an outward facing conformation (4,15,16). Our recent study also demonstrated that a K190C/S mutation from CL1 enhances ATP-independent channel opening induced by a K978C/P/S mutation from CL3 (17). Thus, CLs may function as a key regulatory switch to modulate normal CFTR activity. * This work was supported, in whole or in part, by National Institutes of Health Grant 2R...