Molecular interactions in the pore domain of Na channels

Voltage-gated Na+ channels are pore forming, ion conducting macromolecules which are located in the cell membrane and have a central role in physiology: they enable transmission of electrical signals between cells and, therefore, are essential for physiological processes like the beating of the heart, skeletal muscle motion, and signal transduction in the nervous system. The proper function of Na+ channels requires coordinated gating, i.e. the correct opening and closing of their ion-permeable pores. Upon depolarization of the cell membrane, the channels first open and then, within a few milliseconds, enter a non-conducting state, called fast inactivation. Channels recover from fast inactivation within a few milliseconds of repolarization. On a molecular level, fast inactivation results from an occlusion of the inner vestibule of the pore by an intracellular loop connecting domains III and IV (DIII, DIV). If the membrane remains depolarized for longer periods (i.e. several hundred milliseconds to several minutes) a number of more stable inactivated states are entered. One such state, called "ultra-slow" inactivation (IUS) is extremely long-lasting as it requires 10-20 minutes at hyperpolarized potentials to be removed. Previously, we showed that both the selectivity filter and the internal vestibule of the channel are involved in the generation of I US. Thus, the mutation K1237E in the selectivity filter of the adult rat skeletal muscle Na+ channel (DIII P-loop) strongly favors entry into I US. Recently, we discovered that mutating site 1575 in the DIV S6 segment protected channels carrying the mutation K1237E from entry into IUS. Position 1575 of the DIV S6 segment is predicted to be in close proximity to the selectivity filter, suggesting that I US occurs by an interaction between sites 1237 (selectivity filter) and 1575 (DIV S6 segment). We also showed that the protection from entry into I US is unique to site 1575, as no other mutation in the DIV S6 segment had a similar effect. The current proposal is designed to explore in more detail the nature of the interaction between sites 1237 and 1575. To this end we are planning to exchange the amino acids at these locations by residues of different charge, side chain volume, and hydrophobicity, and to test the effect of such exchanges on I US. We will also investigate to which extent the interaction between sites 1237 and 1575 is affected by the size of the permeating ion. Furthermore, we will explore whether fast inactivation is also modulated by an interaction between the DIV S6 segment and the DIII P-loop. We will apply thermodynamic mutant cycle analysis in order to detect possible coupling between the mutation K1237E in the selectivity filter and serial cysteine and/or alanine replacements in the DIV S6 segment. Finally we will test whether serial mutations in the DIV S6 segment (alone and in combination with the mutation K1237E) modulate the binding behaviour of antidepressant drugs to the internal vestibule. The data will be used to refine a previously published molecular model of the pore of the voltage-gated Na+ channel.

Voltage-gated Na+ channels are pore forming, ion conducting macromolecules which are located in the cell membrane and have a central role in physiology: they enable transmission of electrical signals between cells and, therefore, are essential for physiological processes like the beating of the heart, skeletal muscle motion, and signal transduction in the nervous system. The proper function of Na+ channels requires coordinated gating, i.e. the correct opening and closing of their ion-permeable pores. Upon depolarization of the cell membrane, the channels first open and then, within a few milliseconds, enter a non-conducting state, called fast inactivation. Channels recover from fast inactivation within a few milliseconds of repolarization. On a molecular level, fast inactivation results from an occlusion of the inner vestibule of the pore by an intracellular loop connecting domains III and IV (DIII, DIV). If the membrane remains depolarized for longer periods (i.e. several hundred milliseconds to several minutes) a number of more stable inactivated states are entered. One such state, called "ultra-slow" inactivation (IUS) is extremely long-lasting as it requires 10-20 minutes at hyperpolarized potentials to be removed. Previously, we showed that both the selectivity filter and the internal vestibule of the channel are involved in the generation of I US. Thus, the mutation K1237E in the selectivity filter of the adult rat skeletal muscle Na+ channel (DIII P-loop) strongly favors entry into I US. Recently, we discovered that mutating site 1575 in the DIV S6 segment protected channels carrying the mutation K1237E from entry into I US. Position 1575 of the DIV S6 segment is predicted to be in close proximity to the selectivity filter, suggesting that I US occurs by an interaction between sites 1237 (selectivity filter) and 1575 (DIV S6 segment). We also showed that the protection from entry into I US is unique to site 1575, as no other mutation in the DIV S6 segment had a similar effect. The current proposal is designed to explore in more detail the nature of the interaction between sites 1237 and 1575. To this end we are planning to exchange the amino acids at these locations by residues of different charge, side chain volume, and hydrophobicity, and to test the effect of such exchanges on I US. We will also investigate to which extent the interaction between sites 1237 and 1575 is affected by the size of the permeating ion. Furthermore, we will explore whether fast inactivation is also modulated by an interaction between the DIV S6 segment and the DIII P-loop. We will apply thermodynamic mutant cycle analysis in order to detect possible coupling between the mutation K1237E in the selectivity filter and serial cysteine and/or alanine replacements in the DIV S6 segment. Finally we will test whether serial mutations in the DIV S6 segment (alone and in combination with the mutation K1237E) modulate the binding behaviour of antidepressant drugs to the internal vestibule. The data will be used to refine a previously published molecular model of the pore of the voltage-gated Na+ channel.