Common Principles of Voltage-Dependent Gating for Hv and Kv Channels

Abstract

Voltage-activated proton (Hv1) channels are relatives of classical voltage-activated cation channels. In this issue of Neuron, Hong et al. (2013) and Qiu et al. (2013) investigate the functional mechanisms of Hv1 gating and uncover key relationships with Kv channels.

Voltage-activated ion channels are critical for many fundamental processes in the nervous system. Voltage-activated sodium (Nav) and potassium (Kv) channels are responsible for the generation and propagation of the nerve impulse, and voltage-activated calcium (Cav) channels mediate most forms of excitation-secretion coupling. These classical voltage-activated ion channels are tetramers (Kv channels) or pseudotetramers (Nav and Cav channels), with six transmembrane segments in each subunit that together sense membrane voltage (S1–S4) and form the central pore domain (S5–S6; Figure 1A). For decades, voltage-activated channels were believed to conform to this architecture until the discovery of voltage-activated proton channels (Hv1) (Ramsey et al., 2006; Sasaki et al., 2006), which were subsequently found to be homodimers (Koch et al., 2008; Lee et al., 2008; Tombola et al., 2008), with each monomer consisting of only four segments related to S1–S4 (Figure 1B). Comprehending how S1–S4 acts both as a pore and a voltage sensor has been the focus of intense investigation since Hv1 was discovered (DeCoursey, 2008).

Figure 1. Architecture of Voltage-Activated Cation Channels.

(A) Architecture of classical voltage-activated channels. Kv channels are tetramers, with each subunit containing six segments spanning the membrane (gray slab). S1–S4 from each subunit form a voltage-sensing domain in the tetrameric channel (only one is depicted). S5–S6 from four subunits form the central pore domain. The S4 Arg residues (blue) drive motions of that helix in response to voltage changes. A well-conserved Phe in S2 (green) is the likely charge-transfer center. Acidic residues stabilizing Arg residues within the membrane are not shown. Both Nav and Cav channels contain four repeating S1–S6 segments within a subunit (pseudotetramers).

(B) Architecture of dimeric Hv channels. Each subunit contains four transmembrane segments corresponding to the S1–S4 in Kv channels. Hv1 channels do not contain separate pore domains, and the permeation pathway for protons is contained within S1–S4. Dimerization interfaces exist between S1 helices and in the C-terminal coiled-coil. The S1 helix acidic residue studied by Qiu et al. (2013) is shown in red.

(C) Chemical structures of an Arg side chain and the Hv1 inhibitor 2GBI described by Hong et al. (2013), with guanidine groups (blue).

Understanding mechanisms of opening and closing of any ion channel begins with identifying where the pore (the permeation pathway) and the “gate” (the region that prevents ion flow in the closed state) reside. In this issue of Neuron, Hong et al. (2013) and Qiu et al. (2013) reveal principles of Hv1 that parallel other voltage-activated channels. Hong et al. (2013) tackled locating the pore and gate by identifying new inhibitors and studying their mechanism of action. This harkens back to classical studies of the Armstrong and Yellen laboratories (Armstrong, 1974; Holmgren et al., 1997), where blocking Kv channels by quaternary ammonium ions like tetraethylammonium (TEA) showed that intracellular TEA binds to the pore only after the gate opens and subsequently prevents the gate from shutting, an effect often referred to as “foot in the door.” Hong et al. (2013) sought compounds with a similar effect on Hv1. Reasoning that if S4 Arg residues move through an aqueous pathway within voltage-sensing domains, and guanidinium ions can permeate a Kv channel’s voltage sensor (Tombola et al., 2005), screening guanidinium-containing compounds would be a logical starting point (Figure 1C). Hong et al. (2013) found that guanidinium ions are weak blockers but that compounds containing aromatic substituents on the guanidine scaffold produce robust inhibition (Figure 1C). Applying their best inhibitor (2GBI) to the intracellular side of the membrane when the channel is closed or open, like TEA on Kv channels, Hv1 could not be blocked in the closed state but became rapidly blocked once channels opened with membrane depolarization. 2GBI dramatically slowed channel closing, reminiscent of the foot in the door effect of TEA on Kv channels. Together, this suggests the intracellular end of the pore must open for 2GBI to bind and 2GBI must unbind for the pore to close. Strikingly, mutating a unique position (F150; Figure 1B) in the S2 helix to alanine increases the affinity of 2GBI over 300-fold. This highly conserved aromatic residue is likely the charge-transfer center in voltage sensors, a region that S4 Arg residues traverse when moving outward with depolarization (Tao et al., 2010). The robust enhancement in blocker affinity fits nicely with the blocker tightly snuggling into the void created by the F150A mutation, providing a likely scenario for a relatively local effect on blocker binding. This offers direct evidence for the location of the permeation pathway on the intracellular side of the pore and demonstrates that this region undergoes a conformational change during channel opening.

Investigating recovery from inhibition by 2GBI, Hong et al. (2013) also found that the subunits of Hv1 interact cooperatively. In this experiment, Hv1 is first opened using membrane depolarization in the presence of intracellular 2GBI, leading to blockage of a sizable fraction of the outward H⁺ current. The channel is then closed with hyperpolarization for varying time intervals before re-opening with depolarization. With dimeric channels, a fraction opens more rapidly during the second depolarization, while monomeric channels had no difference in their reopening kinetics. This is suggestive of a population of dimeric channels where one pore was blocked with its gate locked in the open state, and the other was unblocked with its gate closed. With two gates cooperatively coupled, this population of channels opens more rapidly than when both gates were initially closed. When validating this with a model, Hong et al. (2013) invoked an additional constraint that closure of one gate slows dissociation of 2GBI from the neighboring (blocked) subunit, what they term “hemi-channel blocker trapping.” The structural basis of this cooperativity will be an interesting topic for future investigations, and may involve the S1 helices, a potential dimer interface within the membrane, or the initial C-terminal region that forms a coiled-coil critical for dimerization (Fujiwara et al., 2012; Koch et al., 2008; Lee et al., 2008; Tombola et al., 2008).

Qiu et al. (2013) investigate Hv1 gating mechanisms by measuring fluorescence changes of subunits tagged with a fluorophore near S4. They initially observe a decrease in fluorescence with membrane depolarization, similar to changes seen in Kv channels. However, they also observed a relatively rapid further decrease in fluorescence, which they termed the “hook,” when the membrane voltage is subsequently repolarized to its negative resting value. The initial decrease in fluorescence occurred at voltages more negative than required for H⁺ currents and developed more rapidly, suggesting a conformational change in the voltage-sensor preceding pore opening. In contrast, the hook had the same voltage range and similar kinetics required for H⁺ currents, suggesting a conformational change more closely associated with pore opening in Hv1. Qiu et al. (2013) conclude that there must be two types of fluorescence changes following depolarization: the first is a decrease reporting voltage-sensor activation, and the second is an increase reflecting pore opening. The hook is observed because the fluorescence increase reverses rapidly when the membrane is repolarized.

With a similar experiment using monomeric Hv1, channel opening and the hook shift to more depolarized voltages. Mutations of an Asp at the S1-S1 interface (Figure 1B) results in similar shifts, suggesting both the C-terminal coiled-coil interface (disrupted in the monomerized construct) and the S1-S1 interface contribute to cooperative opening. Both of these channel variants have a large separation between the voltage range where the voltage sensors initially activate and where the channels open, reminiscent of ILT mutations in the Shaker Kv channel (Ledwell and Aldrich, 1999). ILT mutations in the S4 helix in Shaker alter the cooperative opening transition to produce a large separation between the voltages where sensors activate and the channel opens. The ILT mutation has been an extremely useful tool for understanding gating of Shaker, and the discovery by Qiu et al. (2013) has similar value. In an upcoming paper, the Larsson group found that a charge-neutralizing mutation of the outermost S4 arginine in Hv1 (R255A) prevents voltage sensors from fully retracting into the resting state at negative voltages but allows fluctuation between activated (but not open) and open states (C. Gonzales, S. Rebolledo, M.E. Perez, and H.P. Larsson, unpublished data). Using this mutation, Qiu et al. (2013) study the final opening transition in isolation and observe only fluorescence increases with membrane depolarization, supporting their interpretation of the hook. They also found that both the kinetics and voltage dependence of these fluorescence changes nicely overlap with those of channel opening. Importantly, the kinetics of fluorescence increase and channel opening can be described with a single exponential function, suggesting the final opening step in Hv1 is highly cooperative. In sum, Qiu et al. (2013) nicely demonstrate that gating of Hv1 involves at least two conformation changes; first as the voltage sensors move from resting to activated states, and second as they move cooperatively between activated and open states.

Several exciting implications emerge from these two papers. First, although Hv1 and Kv channels are architecturally distinct, they share a common mechanistic paradigm with two fundamental steps—an initial conformational change in each voltage sensor that occurs relatively independently, followed by a final opening transition involving cooperative motion of the subunits. With the Shaker Kv channel, the final opening transition involves motions of S1–S4 and the separate pore domain, whereas in Hv1, both types of conformational changes are restricted to the two coupled S1–S4 domains. This conceptually related gating mechanism in both dimeric Hv1 channels and tetrameric Kv channels, presumably evolved to tune the kinetic behavior of the channels for their functions. Second, Hong et al. (2013) demonstrates that Hv1 can be targeted with small molecule inhibitors, providing a crucial starting point to synthesize derivatives of guanidine compounds for therapeutic applications. The recent demonstration of diminished neuronal death after stroke in Hv1 knockout mice provides a compelling potential application for selective Hv1 inhibitors (Wu et al., 2012). Finally, some of the compounds may be useful for crystallizing the Hv1 channel and stabilizing it in the open state. These pharmacological tools and Hv1 mutations serve as valuable additions to the arsenal of ion channel biophysicists and physiologists, to enable further exploration of these intriguing miniature voltage-activated channels.