Voltage-gated channel mechanosensitivity: fact or friction?

Abstract

The heart is a continually active pulsatile fluid pump. It generates appropriate forces by precisely timed and spaced engagement of its contractile machinery. Largely, it makes its own control signals, the most crucial of which are precisely timed and spaced fluxes of ions across the sarcolemma, achieved by the timely opening and closing of diverse voltage-gated channels (VGC). VGCs have four voltage sensors around a central ion-selective pore that opens and closes under the influence of membrane voltage. Operation of any VGC is secondarily tuned by the mechanical state (i.e., structure) of the bilayer in which it is embedded. Rates of opening and closing, in other words, vary with bilayer structure. Thus, in the intensely mechanical environment of the myocardium and its vasculature, VGCs kinetics might be routinely modulated by reversible and irreversible nano-scale changes in bilayer structure. If subtle bilayer deformations are routine in the pumping heart, VGCs could be subtly transducing bilayer mechanical signals, thereby tuning cardiac rhythmicity, collectively contributing to mechano-electric feedback. Reversible bilayer deformations would be expected with changing shear flows and tissue distension, while irreversible bilayer restructuring occurs with ischemia, inflammation, membrane remodeling, etc. I suggest that tools now available could be deployed to help probe whether/how the inherent mechanosensitivity of VGCs - an attribute substantially reflecting the dependence of voltage sensor stability on bilayer structure - contributes to cardiac rhythmicity. Chief among these tools are voltage sensor toxins (whose inhibitory efficacy varies with the mechanical state of bilayer) and arrhythmia-inducing VGC mutants with distinctive mechano-phenotypes.

Keywords: LQT3; arrhythmias; bleb; ectopic excitation; mechano-electric feedback; pacemaker; sodium channel; stretch.

Figure 1

The structural basis of mechanosensitivity in VGCs. (A) Left, a Kv channel (Long et al., 2007) modeled in closed-like and open-like conformations and right (for intracellular and extracellular faces of the Kv) space-filling models of these states (modified from Chanda et al., 2005) and based on (Long et al., 2007) and refs therein). Due to its cruciform shape, the VGC’s lateral interactions with bilayer lipids are considerably more extensive than if the protein were cylindrical (the typical depiction before high resolution structures were available). Because of the substantial shape change of the closed-open transition, some of the free energy of the transition must be attributed to restructuring the lateral protein-lipid interface. (B) The 4 voltage sensors, whose gating charge is primarily in transmembrane segment S4 (asterisk), occupy the periphery (see Long et al., 2007). (C) A single activated-state voltage sensor domain modeled in lipid bilayer (the two water/lipid interfaces are depicted) showing that in its activated-state, the sensor locally deforms/thins the bilayer (modified from Krepkiy et al., 2009). (D) Bilayers, too have different structures (and hence energetics) that are dramatic and lipid-dependent, as seen from these lateral pressure profiles calculated for two simple bilayers, one without, one with cholesterol. Cholesterol thickens a bilayer (see Z axis) and increases its packing order. For any shape-changing integral membrane protein (IMP), the lateral pressure profile is an important component of the energetic landscape (for further explanation see Morris and Juranka, ; Finol-Urdaneta et al., ; Morris, 2011a, b).

Figure 2

Use of Xenopus oocyte cell-attached patch recordings to study MS gating in VGCs. (A) Endogenous “stretch-activated cation channel” activity and heterologously expressed Kv channels (Shaker, fast inactivation removed) exhibiting what a naive observer could reasonably construe to be “stretch-activated K channel” activity. In reality it is stretch-modulated VGC current (Gu et al., 2001). (B) A Xenopus oocyte, and a cartoon of pipette aspiration as used for applying membrane stretch (inset, a cell-attached macropatch; typically, smaller patches are used but this allows for visualization of non-traumatized plasma membrane; inset is modified from Shcherbatko et al., 1999). As explained in refs (Morris et al., 2006) and (Wang et al., 2009), membrane trauma, when it happens, is submicroscopic. (C) During pipette aspiration, stretch increases membrane tension, and does so whether aspiration pressure is negative (“suck”) or positive (“blow”), as seen for two very different VGCs, a Kv3 homotetramer and a Nav1.5 pseudotetramer. Multivalent lanthanide ions included in the pipette inhibit endogenous stretch channel activity (and, as expected, right-shift VGC currents by tens of mV). Here, and throughout, black, red, gray traces signify before, during, after stretch. (Di,ii) Illustrates “stretch difference currents” obtained from before/during/after records (two step depolarizations and one ramp depolarization are used here) and demonstrates that the magnitude of the stretch difference currents increase with increasing stretch intensity, while (iii) shows that stretch increases unitary K currents frequency at 0 mV (which corresponds to the reversal potential of endogenous stretch-activated cation channels). These figures are modified from (Gu et al., ; Laitko et al., ; Morris and Juranka, 2007a).

Figure 3

VGC operation is inherently mechanosensitive (MS). (A) Nav1.5 channel current accelerates reversibly with stretch; activation and inactivation rates both speed to the same extent (Gmax has been reached at −30 mV in this patch, so peak I_Na is unaffected by stretch, but activation and fast inactivation both reversibly accelerate; modified from Morris and Juranka, 2007a). (B) a Nav1.5 activation Boltzmann left-shifts reversibly with stretch. (C) Shaker (Kv1) and Shaker-ILT have different rate-limiting voltage transitions in the activation pathway. Stretch affects both, but it accelerates the (largely independent) movement of the Shaker voltage sensors, whereas it decelerates (the late concerted) movement of the voltage sensor in Shaker-ILT, as seen here for voltages near the foot and head of the respective G(V)s; in keeping with the acceleration vs deceleration, stretch left-shifts the Shaker Boltzmann and right-shifts the Shaker-ILT Boltzmann (not shown; see Reference Laitko et al., 2006). (D) In Shaker 5aa, voltage-dependent gating is two orders of magnitude slower than in Shaker (compare to C; note the different time scales), but since an independent (though sluggish) voltage-dependent transition is rate-limiting for activation, this mutant channel behaves like its “WT” counterpart (see Laitko and Morris, 2004). (E) HCN2 channels open with hyperpolarization, they close with depolarization, they have multiple open states and they exhibit pronounced hysteresis. Here HCN2 currents are studied by (i) ramp clamp, (ii) passive action potential clamp and (iii) classic step clamp (see Lin et al., 2007) for details. Upon a step depolarization (iii) from −140 to −40 mV, stretch accelerates channel closure. Gating currents show this transition to be analogous to the rate-limiting depolarization-induced “outward” voltage sensor motion in Kv Shaker.

Figure 4

Membrane trauma can irreversibly left-shift Nav channel operation, producing “leaky” Nav channels, probably because the resident bilayer gets more bleb-like, i.e., thinner, more disordered. (A) This phenomenon was first discovered from accidental damage to membranes while making gigaohm seals on oocytes. Here, Nav1.4 (without the auxiliary beta subunit) kinetics recorded in 4 patches from the same oocyte look radically different depending on the extent to which the membrane was mechanically traumatized during seal formation. Modifed from (Tabarean et al., 1999). (B) Cartoon suggesting that bleb formation leaves channels (dark ovals) in a bilayer environment that is on average thinner and (not specifically depicted but indicated by the lighter color) more disordered/fluid/symmetric-across-leaflets. In oocytes, Nav1.5 channels behave as if they are trafficked to disordered membrane (Morris and Juranka, ; Wang et al., ; Banderali et al., 2010) and so do not show irreversible kinetic changes with stretch, but in HEK cells, stretch is largely traumatic for Nav1.5 channels (Beyder et al., 2010). (C) The trauma phenomenon also occurs for Nav1.6 in oocyte membranes and is summarized by coupled left-shift of the activation and steady-state inactivation curves as shown here (see Wang et al., 2009).

Figure 5

Are there more MS voltage sensor toxins out there? (A) A collection of ICK peptides purified from tarantula venom (modified from Jung et al., 2005). The top 3 are established voltage sensor toxins, and the bottom one, GsMTx4, might be too (micromolar level GsMTx4 inhibits unidentified MS cation channels in astrocytes, but that is not the effect of interest here. GsMTx4 has powerful cardiac actions at <200 nm; Bode et al., 2001). (B) The “silver bullet scenario” as described in the text. VsTx1 action is mechanosensitive in that it inhibits this species of Kv channel (right-shifts activation; ii → iii) only when bilayer organization has been disrupted. The irreversible left-shift (i → ii) upon excision/trauma is as seen for some Nav channels (Figure 4), and like the advent of toxin efficacy, is ascribed to, (C) the difference in bilayer mechanics between the two recording conditions. (D) how GsMTx4 might act as a stretch-sensitive toxin for HCN channel voltage sensors. The scenario at left mirrors known behavior of VsTx1 and SGTx on their targets (Kv voltage sensors) and that at right suggests how membrane stretch might increase the inhibitory efficacy of GsMtx4. It is also possible to imagine membrane deformations that decreased the affinity of amphiphilic voltage sensor toxins for their targets. HCN channels are mentioned here, but the idea would apply for any voltage sensor, even for stand-alone ones (see Figure 1C; proton channels, voltage sensitive phosphatases as described, e.g., in Krepkiy et al., 2009).

Figure 6

Two LQT3 channels: impaired domain-4 voltage sensors, impaired mechanosensitivity. (A) Stretch difference currents for WT Nav1.5 channels (see Figure 3A for I_Na before, during, after stretch; Morris and Juranka, 2007a). Because I_Na is inward over the entire physiological range, and because Nav channels activate then rapidly inactivate, stretch difference currents for Nav channels are more complex than stretch difference currents for Kv channels (Figure 2D). For Nav rule is: downward = more I_Na during stretch, upward = less I_Na during stretch. Based on the Nav stretch difference currents at the voltages illustrated, imagine what would happen during an action potential: briefly near the start, stretch would increase I_Na (downward) but quickly decreased I_Na would prevail. If this happens in situ, when there is stretch, the faster turn off of I_Na once an action potential was triggered would contribute in an anti-arrhythmic fashion to the overall mechano-electric feedback. The LQT3 mutations, R1623Q and R1626P, affect the Nav domain 4 voltage sensor, whose depolarization-induced repacking renders the inactivation rate (Tau⁻¹) voltage dependent by stabilizing the inactivation particle in its bound state (see Banderali et al., 2010). Replacing arginine (positive) at R1623 with glutamine (neutral) impairs repacking of the voltage sensor protein and reduces the voltage sensitivity of Tau inactivation. Replacing arginine at 1626 with kink-inducing proline makes the inactivation rate entirely insensitive to voltage; at all voltages, R1626P has the same low-speed inactivation. The consequence: these LQT3 Nav channels remain open too long during the action potential; excess I_Na causes “torsades de pointes” arrhythmias. (B) Stretch (from pipette suction) is less effective at speeding the rate (Tau⁻¹) of I_Na inactivation for R1623Q than for WT (likewise for R1626P, but not shown). (C) Overlaid stretch difference currents compare kinetics for WT and the LQT3 mutant. With increasing depolarization (imagine the action potential passing through −30 and 0 mV) WT I_Na turns off (goes upward) far sooner than for R1623Q. (D) For R1626P, stretch difference current always (even at 0 mV) represents excess (stretch-induced) inward I_Na. The R1626P response to stretch in situ, were it to occur, would be distinctly “excitatory” and pro-arrhythmic. Figures B–D are from Banderali et al. (2010).