Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov;167(6):1378-88.
doi: 10.1111/j.1476-5381.2012.02092.x.

Verapamil- and state-dependent effect of 2-aminoethylmethanethiosulphonate (MTSEA) on hK(v)1.3 channels

Azadeh Nikouee  1 , Malika Janbein, Stephan Grissmer
Affiliations

Verapamil- and state-dependent effect of 2-aminoethylmethanethiosulphonate (MTSEA) on hK(v)1.3 channels

Azadeh Nikouee et al. Br J Pharmacol. 2012 Nov.

Abstract

Background and purpose: T-cells usually express voltage-gated K(v) 1.3 channels. These channels are distinguished by their typical C-type inactivation. Therefore, to be able to rationally design drugs specific for the C-type inactivation state that may have therapeutic value in autoimmune disease therapy, it is necessary to identify those amino acids that are accessible for drug binding in C-type inactivated channels.

Experimental approach: The influence of 2-aminoethylmethanethiosulphonate (MTSEA) on currents through wild-type human K(v)1.3 (hK(v)1.3) and three mutant channels, hK(v)1.3_L418C, hK(v)1.3_T419C and hK(v)1.3_I420C, in the closed, open and inactivated states was investigated by the patch-clamp technique.

Key results: Currents through hK(v)1.3_L418C and hK(v)1.3_T419C channels were irreversibly reduced after the external application of MTSEA in the open state but not in the inactivated and closed states. Currents through hK(v)1.3_I420C channels were irreversibly reduced in the open and inactivated states but not in the closed state. In the presence of verapamil, the MTSEA modification of hK(v)1.3_T419C and hK(v)1.3_I420C channels was prevented, while the MTSEA modification of hK(v)1.3_L418C channels was unaffected.

Conclusion and implications: From our experiments, we conclude that the activation gate of all mutant channels must be open for modification by MTSEA and must also be open during inactivation. In addition, the relative movement of the S6 segments that occur during C-type inactivation includes a movement of the side chains of the amino acids at positions 418 and 419 away from the pore lining. Furthermore, the overlapping binding site for MTSEA and verapamil does not include position 418 in hK(v) 1.3 channels.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Effect of externally applied 1 mM MTSEA on current through (A) hKv1.3 wt channels, (B) hKv1.3_L418C, (C) hKv1.3_T419C and (D) hKv1.3_I420C mutant channels in the closed and open states. The experiments were independently repeated five times (n= 5). (A–D, left) Representative whole-cell currents through the channels were elicited by 200 ms voltage pulses from −120 to +40 mV every 30 s (wt) or every 60 s (mutant channels). The currents were recorded before (trace a) and after adding 1 mM MTSEA (traces b-d) to the bath solution. Between traces a and b, the current recording was stopped and the membrane potential was held for 2 min at −120 mV. (A–D, right) The peak current amplitudes of the elicited currents from the experiment on the left were plotted against the recording time. The letters above the data points refer to the current traces shown on the left. The red lines are fits of exponential functions to the data points yielding time constants of peak current loss in the presence of MTSEA of 102 s (L418C), 78 s (T419C) and 68 s (I420C). HP, holding potential.
Figure 2
Figure 2
Effect of externally applied 1 mM MTSEA on the inactivated state of hKv1.3_L418C, hKv1.3_T419C and hKv1.3_I420C mutant channels. The experiments were independently repeated five times (n= 5). (A–F, left) Representative whole-cell currents were elicited by 200 ms voltage pulses from −120 to +40 mV every 60 s. After the black trace, the membrane potential was adjusted to −20 mV to ensure that all the mutant channels were in the inactivated state and a depolarizing step from this holding potential (HP) to +40 mV was applied (blue trace). After recording this trace, the solution was changed to Na-Ri (A,C,E) or MTSEA (B,D,F). After 2 min, the bath solution was replaced by Na-Ri and the membrane potential was adjusted to −120 mV again from which the next depolarizing voltage step to +40 mV was elicited (red trace). (A–F, right) Peak currents of the recorded traces shown on the left plotted against the recording time. Corresponding changes in the HP and solution changes are given in the boxes on top.
Figure 3
Figure 3
Possible protection against the effect of 1 mM MTSEA by the application of 100 µM verapamil (VP) to the bath solution in hKv1.3_L418C, hKv1.3_T419C and hKv1.3_I420C mutant channels. The experiments were independently repeated five times (n= 5). (A–C, left) Representative whole-cell currents were elicited by 200 ms depolarizing voltage steps from a holding potential (HP) of −120 to +40 mV every 60 s in different solutions as can be seen on the right side of the graph. (A–C, right) Peak current amplitudes of the currents elicited in the experiment on the left were plotted against the recording time. After having recorded three control traces in a bath solution containing Na-Ri, the bath solution was replaced several times: firstly, by a Na-Ri solution containing 100 µM VP; secondly, by a Na-Ri solution containing 100 µM VP + 1 mM MTSEA (MTSEA + VP); thirdly, by a Na-Ri solution containing 100 µM VP; and finally, by a Na-Ri solution as in the beginning (Na-Ri) of the experiment. The letters above the points refer to the current traces shown on the left.
Figure 4
Figure 4
Side view of two monomers of the Kv1.3 channel highlighting the amino acid residues in the α-helix of a single S6 segment that were mutated to cysteines. For reasons of clarity, just S5–S6 of each monomer is shown in this picture. The inset highlights the close-up view of residues in S6 that were substituted to cysteines in our experiments. Molecular graphic images were produced by using VMD1.8.7 software.
Figure 5
Figure 5
Docking of verapamil into a homology model of the hKv1.3 channel. The representative hKv1.3 channel structure is composed of two monomers and each monomer includes S5, S6; and the P-turn is shown in grey. The inset highlights the close-up of the positions of verapamil and residues in S6, which were substituted in our experiment. The residues in one monomer have been shown in different colours (V417: red, L418: orange, T419: green, I420: magenta).
Figure 6
Figure 6
Docking of MTSEA into the hKv1.3_L418C mutant channel in the presence of verapamil. Two monomers of the channel are shown in grey, position 418 is shown in orange. The inset highlights the close-up view of the positions of verapamil, MTSEA (yellow) and residue C418 (orange) in S6.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Choi KL, Aldrich RW, Yellen G. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc Natl Acad Sci USA. 1991;88:5092–5095. - PMC - PubMed
    1. Cordero-Morales JF, Cuello LG, Zhao Y, Jogini V, Cortes DM, Roux B, et al. Molecular determinants of gating at the potassium-channel selectivity filter. Nat Struct Mol Biol. 2006;13:311–318. - PubMed
    1. Cuello LG, Jogini V, Cortes DM, Perozo E. Structural mechanism of C-type inactivation in K+ channels. Nature. 2010a;446:203–209. - PMC - PubMed
    1. Cuello LG, Jogini V, Cortes DM, Pan AC, Gagnon DG, Dalmas O, et al. Structural basis for the coupling between activation and inactivation gates in K+ channels. Nature. 2010b;466:272–275. - PMC - PubMed
    1. DeCoursey TE. Mechanism of K+ channel block by verapamil and related compounds in rat alveolar epithelial cells. J Gen Physiol. 1995;106:745–779. - PMC - PubMed

Publication types