hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state

Abstract

Delayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5-S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5 L ) to the channel S6 C-terminus (S6 T ) completely inhibits hERG. This shows that channel S4-S5 L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6 T to the channel S4-S5 L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5 L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5 L acts as a voltagecontrolled ligand that binds S6 T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltagegated channels.Voltage-dependent ion channels are ubiquitously expressed in human tissues. They perform a plethora of physiological functions such as generation and modulation of the electrical activity in excitable tissues, modulation of neurotransmitter and hormone release, and electrolyte transport in epithelia. Canonical voltage-gated ion channels are tetramers of subunits containing six transmembrane segments (S1 to S6). Each of the four subunits is composed of one voltage sensor domain (S1 to S4) and a pore domain (S5-S6). The four pore domains tetramerize to generate a single pore module, which is regulated by the four voltage sensor domains 1, 2 . One key question remains: how do the voltage sensor domains regulate pore gating? Several independent approaches support the idea that interaction between the S4-S5 linker (S4-S5 L ) and the S6 C-terminal (S6 T ) part plays a critical role in that regulation [2][3][4][5] . First, Lu and collaborators pinpointed the sequence complementarity between S4-S5 L and S6 T in the Shaker channel 4,5 . Second, structural data indicated the close proximity between S4-S5 L and S6 T and suggested a mechanical lever model to explain the coupling between the voltage sensor and the gate of the related K v 1.2 channel 2 . However, the mechanism may prove to be more complex. Since 2006, several works on other eukaryotic K v channels suggested state-dependent S4-S5 L and S6 T interactions 3,[6][7][8][9][10] , but the precise mechanism remains elusive and may vary from one channel to another.In a previous study, we suggested that the voltage dependence of the cardiac voltage-gated KCNQ1 channel (K v 7.1) follows a ligand/receptor model. In this model, S4-S5 L is a ligand whose interaction with S6 T is only possible at resting potentials, and this interaction locks the channel in a closed s...

Up-regulation of voltage-gated sodium channels by peptides mimicking S4-S5 linkers reveals a variation of the ligand-receptor mechanism

“…When the membrane is depolarized, S4 pulls S4-S5 L out of its binding pocket, leading to channel opening. This is consistent with the observation that specific S4-S5 L -mimicking peptides inhibit hK V 7.1 and hK V 11.1 channels, by replacing the endogenous segment in the binding pocket 25,26 . This mechanism was recently extended to hK V 10.2 channels 29 .…”

“…We identified one activating S4-S5 L peptide in Na V Sp1 and four in hNa V 1.4, suggesting that Na V channels follow a ligand/ receptor model of voltage-dependent gating (Fig. 11c): when the membrane is depolarized, endogenous S4-S5 L stabilizes the open state of Na V channels, as indicated by the Na V Ms structure captured in the open state 8,16,17 . This contrasts with what is happening with K V channels: when the membrane is polarized, endogenous S4-S5 L stabilizes the closed state of K V channels, as suggested by several studies 25,26,29 .…”

“…In this work, we used a S4-S5 L mimicking peptide approach to test whether voltage-gated sodium channels follow the ligand/receptor model previously proposed for hK V 7.1 25 , hK V 11.1 26 and hK V 10.2 29 channels. We identified one activating S4-S5 L peptide in Na V Sp1 and four in hNa V 1.4, suggesting that Na V channels follow a ligand/ receptor model of voltage-dependent gating (Fig.…”

“…Such allosteric regulation has been suggested for several channels, including hK V 11.1 (hERG) and hK V 7.1 (KCNQ1) channels 23,24 . In these channels, we elucidated the nature of this allosteric coupling: when S4 sensors are in the "down" or deactivated conformation, the four S4-S5 L bind to S6 T in the closed state, stabilizing this state 25,26 . Noteworthy, ATP has also been shown to stabilize the closed state of K ATP channels 27,28 .…”

“…From the interaction motif observed in Na V Ms channel 8,16,17 , we hypothesized that S4-S5 L acts as a ligand binding to S6 T and stabilizing the channel open-state and not the closed state. We used the same peptide approach previously used for hK V 7.1, hK V 11.1 and hK V 10.2 channels 25,26,29 to test whether S4-S5 L peptides lead to a gain of function in both prokaryotic and eukaryotic Na V channels.…”