PDBsum entry 1eoe

Membrane protein

PDB id: 1eoe

Contents

Protein chain

100 a.a. *

Waters ×103

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Voltage dependent activation of potassium channels is coupled to t1 domain structure.

Authors

S.J.Cushman, M.H.Nanao, A.W.Jahng, D.Derubeis, S.Choe, P.J.Pfaffinger.

Ref.

Nat Struct Biol, 2000, 7, 403-407. [DOI no: 10.1038/75185]

PubMed id

10802739

Abstract

The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.

Figure 1.
Figure 1. Location of T1 domain point mutations. A model of a Kv channel1 is depicted based on known structures. Only two subunits on opposite sides of the Kv channel are shown for clarity. The T1 domain2, 3 is shown to scale below the TM pore region, which is depicted based on the structure of the KcsA channel4. An N-terminal inactivation ball structure^1, based on the NMR structure of the Shaw type Kv channel Kv3.4 (Raw3) inactivation ball44 is shown to scale connected to one of the T1 subunits. Regions for which no structural information is available are given as black linking segments (segment A links the N-terminal inactivation ball to the T1 domain; B is the T1 to S1 linker; C is the S4 to S5 linker and segment D links the S6 to the C-terminus). S1 -S6 are the proposed Kv, only S5 and S6 are modeled here. The four-fold symmetry axis at the center of the hypothetical Kv channel is vertical and indicated by arrow 1. The backbone conformation of the T1 subunit chain is shown in color gradually changing from blue (N-terminus) to white (C-terminus). The model depicts a 'right side up' relationship between the T1 domain and the transmembrane domains. An 'upside down' relationship would have the darker N-terminal regions of the T1 domain near the TM pore region. The two side chains where mutations have been introduced are highlighted: Val 135, blue; Asn 136, green. Potential pathways for ions to reach the TM pore are between the T1 domain and the TM pore (arrow 2) or up the aqueous central cavity at the axis of symmetry (arrow 1). The narrowest part of the T1 cavity along the four fold axis is formed by Asn 136 (green). This figure was prepared using Setor45.

Figure 3.
Figure 3. Analysis of T1 domain tetramer stability. a, Fractional elution of Aplysia Kv1.1 wild type untagged N-terminal cytoplasmic domains from immobilized tetramers, formed by coassembly with otherwise identical His[6]-tagged protein. Tetramers were immobilized by binding of the His[6] squence to metal affinity resin. Elutions were performed under constant flow using standard buffer conditions at 4 °C (blue circle) and at 22 °C (red square). Curves are exponential decay fits to the data, with one exponential term for 4 °C and two exponential terms for 22 °C. The time constant is 5 h n 3 s.e.m. for all data points. b, Dissociation of monomers in 3 M urea. Data were best fit with three exponentials with time constants ranging from 2 min to 4 h at 22 °C (red square) and 9 min to 7 h at 4 °C (blue circle). n 3 s.e.m for all data points. c, Stability of mutant T1 tetramers as determined by urea denaturation. The unfolding free energy of wild type, V135R, N136A and N136D mutants are estimated to be 11.1, 10.2, 9.8 and 7.4 kcal mol-1, respectively. d, Correlation of the energetic effects of T1 mutations on channel gating properties with the change in unfolding free energy of the T1 domain. The functional G for the change in activation midpoint is proportional to the shift in V[1/2] by the factor z[G]F, where z[G] is the gating charge that is moved across the lipid bilayer during channel activation gating and F is Faraday constant. The functional G is proportional to ln ( [mutant]/ [wildtype]) by the factor -RT, for the changes in gate closing time measured at -50 mV. Slopes and intercepts of the regression lines are: 2.2 mV kcal-1 mol-1 and -24.56 mV for the change in half activation; and -0.18 kcal-1 mol-1 and 2.05 mV for the change in ln ( [mutant]/ [wildtype]) measured at -50 mV.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2000, 7, 403-407) copyright 2000.