Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine the extent of this transmembrane movement, we use fluorescent resonance energy transfer between the S4 domain and a reference point in the lipid bilayer. The lipophilic ion dipicrylamine distributes on either side of the lipid bilayer depending on the membrane potential, and is used here as a resonance-energy-transfer acceptor from donor molecules attached to several positions in the Shaker K+ channel. A voltage-driven transmembrane movement of the donor should produce a transient fluorescence change because the acceptor also translocates as a function of voltage. In Shaker K+ channels no such transient fluorescence is observed, indicating that the S4 segment does not translocate across the lipid bilayer. Based on these observations, we propose a molecular model of voltage gating that can account for the observed 13e gating charge with limited transmembrane S4 movement.

The development of genetically encoded fluorescent voltage probes is essential to image electrical activity from neuronal populations. Previous green fluorescent protein (GFP)-based probes have had limited success in recording electrical activity of neurons because of their low sensitivity and poor temporal resolution. Here we describe a hybrid approach that combines a genetically encoded fluorescent probe (membrane-anchored enhanced GFP) with dipicrylamine, a synthetic voltage-sensing molecule that partitions into the plasma membrane. The movement of the synthetic voltage sensor is translated via fluorescence resonance energy transfer (FRET) into a large fluorescence signal (up to 34% change per 100 mV) with a fast response and recovery time (0.5 ms). Using this two-component approach, we were able to optically record action potentials from neuronal cell lines and trains of action potentials from primary cultured neurons. This hybrid approach may form the basis for a new generation of protein-based voltage probes.

Mechanotransduction, the conversion of mechanical stimuli into electrical signals, is a fundamental process underlying essential physiological functions such as touch and pain sensing, hearing, and proprioception. Although the mechanisms for some of these functions have been identified, the molecules essential to the sense of pain have remained elusive. Here we report identification of TACAN (Tmem120A), an ion channel involved in sensing mechanical pain. TACAN is expressed in a subset of nociceptors, and its heterologous expression increases mechanically evoked currents in cell lines. Purification and reconstitution of TACAN in synthetic lipids generates a functional ion channel. Finally, a nociceptor-specific inducible knockout of TACAN decreases the mechanosensitivity of nociceptors and reduces behavioral responses to painful mechanical stimuli but not to thermal or touch stimuli. We propose that TACAN is an ion channel that contributes to sensing mechanical pain.

Background: Although powerful, single subunit counting is time-consuming, prone to user bias, and largely restricted to Xenopus expression. Results: PIF is an automated analysis program that identifies subunit stoichiometry of any fluorescently tagged membrane protein from TIRF recordings. Conclusion: PIF is accurate to more than 90% even in noisy data typical for mammalian expression system. Significance: The PIF approach is generalizable to any membrane protein and TIRF microscope.

Atomic-scale models on the gating mechanism of voltage-gated potassium channels (Kv) are based on linear interpolations between static structures of their initial and final state derived from crystallography and molecular dynamics simulations, and, thus, lack dynamic structural information. The lack of information on dynamics and intermediate states makes it difficult to associate the structural with the dynamic functional data obtained with electrophysiology. Although voltage-clamp fluorometry fills this gap, it is limited to sites extracellularly accessible, when the key region for gating is located at the cytosolic side of the channels. Here, we solved this problem by performing voltage-clamp fluorometry with a fluorescent unnatural amino acid. By using an orthogonal tRNAsynthetase pair, the fluorescent unnatural amino acid was incorporated in the Shaker voltage-gated potassium channel at key regions that were previously inaccessible. Thus, we defined which parts act independently and which parts act cooperatively and found pore opening to occur in two sequential transitions.Anap | two-color VCF V oltage-gated potassium channels (K V ) are essential for generating action potentials in the central nervous system and, when defective, are linked to severe familial diseases including cardiac arrhythmias and epilepsy. The voltage-sensing domains (VSD) of K V channels (transmembrane helices S1-S4; Fig. 1A) undergo a major conformational change upon membrane depolarization driven by the positive charges in the S4, which finally leads to opening of the pore domain (transmembrane helices S5-S6). Based on the consensus on the closed (initial) and open (final) state structures (1-6), the gating movement has been predicted; the S4 helix is projected to slide upward and tilt with respect to the membrane normal, and this movement pushes the S4-S5 linker and the S6 helix inward and closes the ion-conducting pore. However, this projection relies on linear interpolations between the closed and open state and lacks any information on dynamics or intermediate states.As a result, the projected movement does not suffice to explain fundamental characteristics of voltage sensor and pore domain kinetics, detected as "gating" and "ionic" currents, respectively. Such functional electrophysiology measurements revealed that, first, at least one intermediate state has to exist during voltage sensor movement (7) and that, second, voltage sensor movement and pore opening do not occur simultaneously. Each channel consists of four voltage sensors controlling a single central pore. It is thought that the four voltage sensors activate independently, and only after all four have activated, the central pore opens cooperatively (8,9). This mechanism implies that the energy generated by the movement of the first three voltage sensors has to be "conserved" in the system and has to be released to the pore during opening (10). The linear interpolations between closed and open structures leave the basis of both cooperativity and energy conservation un...

LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators. These regulate the immune response, but may also cause severe harm leading to septic shock. The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14. As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior. Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca2+- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages. The channel is activated by endotoxically active LPS in a dose-dependent manner. Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs. Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K+ channel blockers.

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K ؉ channels, the gating currents are asymmetric; although the on-gating currents are fast, the off-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between Arg-394, Glu-395, and Leu-398 on the C termini of the S4-S5 linker and Tyr-485 on the S6 of the neighboring subunit, which is responsible for the development of the slow off-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.The voltage dependence of ion channels is the basis for all electrical signaling in the central nervous system. In tetrameric voltage-gated K ϩ channels, each subunit is composed of six transmembrane ␣-helices (S1-S6), with S1-S4 forming the voltage sensing domain and S5-S6 of all four subunits forming the pore. The voltage-sensing domains are covalently connected to the S5 of the pore region by the S4-S5 linker. The intracellular gate is made up of the S6 C-terminal ends that cross each other, forming a bundle that occludes the pore when the channel is closed. Pore opening in voltage-gated K ϩ channels is controlled by the movement of the voltage sensor in which charged residues of the S4 respond to changes in membrane potential. During this conformational change, the charges are moved through the electric field, generating the transient gating currents (for review, see Ref. 1). Gating currents were first predicted by Hodgkin and Huxley and were first detected in sodium channels by Armstrong et al. (2, 3). The movement is transferred to the pore domain (electromechanical coupling) and subsequently leads to pore opening. Voltage sensor movement precedes pore opening so that the transitions the channel undergoes during electromechanical coupling are reflected in the gating currents.Activation (on) and deactivation (off) gating currents for the non-conducting Shaker-IR channel, W434F (4 -6) have been previously described (6 -8). Briefly, on-gating currents rise and decay quickly after small depolarizations but rise more slowly and exhibit more prolonged and complex decay kinetics after intermediate depolarizations and, finally, develop and decay rapidly after depolarizations large enough to activate all channels. In contrast, offgating currents, which de...

The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region.We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.