Catherine E. Morris

Mechanosensitive ion channels

Cells are seldom static. They grow, migrate, contract, change volume and shape, reorganize internally, divide, and are subject to all manner of compression, shearing and stretch. Evidence is growing that most cells have ion channels potentially capable of monitoring and regulating active and passive variations in cellular mechanics. The ion channels in question are mechanosensitive (MS), that is, their open probability (Pop) depends on stress at the membrane (see Fig. 1 for examples). Channels of this ilk were postulated decades ago as a way to account for mechanoelectrical transduction in muscle spindles, crustacean stretch-receptors, Pacinian corpuscles and other specialized mechanoreceptors [see references in 17, 41-45]. What was not anticipated was the picture now emerging, with MS channels present as membrane components in ordinary pedestrian cells, rather than being confined to the exotic mechanoreceptor specialists. As documented in other recent reviews [27, 44, 49], MS channels of various ionic selectivities have been found in spheroplasts prepared from bacteria and fungi, in protoplasts prepared from plants, and in a multitude of animal cells of vertebrate and invertebrate origin. Presently, the classification MS, which encompasses stretch-activated (SA) and stretch-inactivated (SI) [38] and possibly other channels (Table 1), is merely phenomenological; procaryotic and eucaryotic MS channels, in particular, may be quite unrelated. Commonality of gating mode need not signify a common underlying mechanism. The fundamental biophysical question about MS channels--how does mechanical disturbance of

Membrane Tension in Swelling and Shrinking Molluscan Neurons

When neurons undergo dramatic shape and volume changes, how is surface area adjusted appropriately? The membrane tension hypothesis—namely that high tensions favor recruitment of membrane to the surface whereas low tensions favor retrieval—provides a simple conceptual framework for surface area homeostasis. With membrane tension and area in a feedback loop, tension extremes may be averted even during excessive mechanical load variations. We tested this by measuring apparent membrane tension of swelling and shrinkingLymnaea neurons. With hypotonic medium (50%), tension that was calculated from membrane tether forces increased from 0.04 to as much as 0.4 mN/m, although at steady state, swollen-cell tension (0.12 mN/m) exceeded controls only threefold. On reshrinking in isotonic medium, tension reduced to 0.02 mN/m, and at the substratum, membrane invaginated, creating transient vacuole-like dilations. Swelling increased membrane tension with or without BAPTA chelating cytoplasmic Ca2+, but with BAPTA, unmeasurably large (although not lytic) tension surges occurred in approximately two-thirds of neurons. Furthermore, in unarborized neurons voltage-clamped by perforated-patch in 50% medium, membrane capacitance increased 8%, which is indicative of increasing membrane area. The relatively damped swelling–tension responses ofLymnaea neurons (no BAPTA) were consistent with feedback regulation. BAPTA did not alter resting membrane tension, but the large surges during swelling of BAPTA-loaded neurons demonstrated that 50% medium was inherently treacherous and that tension regulation was impaired by subnormal cytoplasmic [Ca2+]. However, neurons did survive tension surges in the absence of Ca2+ signaling. The mechanism to avoid high-tension rupture may be the direct tension-driven recruitment of membrane stores.

Successive openings of the same acetylcholine receptor channel are correlated in open time.

Previous analysis of single-channel current records has shown that both the opening and closing transitions of chemically activated ion channels are operated by fast and slow kinetic processes. The fast component in the kinetics of channel opening has been interpreted as the reopening of a channel that has just closed. The fast component in the kinetics of channel closure has many possible explanations and is therefore more difficult to interpret. We can gain insight into the closing process by asking whether the lifetimes of successive openings of an acetylcholine receptor channel are correlated in open-state lifetime. Five kinetic models of channel closure are considered. Two of these models predict uncorrelated open-state lifetimes, one predicts correlated open-state lifetimes, and for two others a range of behavior is possible. Acetylcholine receptor channel data from cultured rat muscle are analyzed to show that open-state lifetimes are correlated, eliminating two models of channel gating.

Mechanosensitivity of N-Type Calcium Channel Currents

Mechanosensitivity in voltage-gated calcium channels could be an asset to calcium signaling in healthy cells or a liability during trauma. Recombinant N-type channels expressed in HEK cells revealed a spectrum of mechano-responses. When hydrostatic pressure inflated cells under whole-cell clamp, capacitance was unchanged, but peak current reversibly increased ~1.5-fold, correlating with inflation, not applied pressure. Additionally, stretch transiently increased the open-state inactivation rate, irreversibly increased the closed-state inactivation rate, and left-shifted inactivation without affecting the activation curve or rate. Irreversible mechano-responses proved to be mechanically accelerated components of run-down; they were not evident in cell-attached recordings where, however, reversible stretch-induced increases in peak current persisted. T-type channels (alpha(1I) subunit only) were mechano-insensitive when expressed alone or when coexpressed with N-type channels (alpha(1B) and two auxiliary subunits) and costimulated with stretch that augmented N-type current. Along with the cell-attached results, this differential effect indicates that N-type mechanosensitivity did not depend on the recording situation. The insensitivity of T-type currents to stretch suggested that N-type mechano-responses might arise from primary/auxiliary subunit interactions. However, in single-channel recordings, N-type currents exhibited reversible stretch-induced increases in NP(o) whether the alpha(1B) subunit was expressed alone or with auxiliary subunits. These findings set the stage for the molecular dissection of calcium current mechanosensitivity.

Membrane Stretch Affects Gating Modes of a Skeletal Muscle Sodium Channel

The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension.

Voltage-Gated Channel Mechanosensitivity: Fact or Friction?

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.

Membrane Tension Accelerates Rate-limiting Voltage-dependent Activation and Slow Inactivation Steps in a Shaker Channel

A classical voltage-sensitive channel is tension sensitive—the kinetics of Shaker and S3–S4 linker deletion mutants change with membrane stretch (Tabarean, I.V., and C.E. Morris. 2002. Biophys. J. 82:2982–2994.). Does stretch distort the channel protein, producing novel channel states, or, more interestingly, are existing transitions inherently tension sensitive? We examined stretch and voltage dependence of mutant 5aa, whose ultra-simple activation (Gonzalez, C., E. Rosenman, F. Bezanilla, O. Alvarez, and R. Latorre. 2000. J. Gen. Physiol. 115:193–208.) and temporally matched activation and slow inactivation were ideal for these studies. We focused on macroscopic patch current parameters related to elementary channel transitions: maximum slope and delay of current rise, and time constant of current decline. Stretch altered the magnitude of these parameters, but not, or minimally, their voltage dependence. Maximum slope and delay versus voltage with and without stretch as well as current rising phases were well described by expressions derived for an irreversible four-step activation model, indicating there is no separate stretch-activated opening pathway. This model, with slow inactivation added, explains most of our data. From this we infer that the voltage-dependent activation path is inherently stretch sensitive. Simulated currents for schemes with additional activation steps were compared against datasets; this showed that generally, additional complexity was not called for. Because the voltage sensitivities of activation and inactivation differ, it was not possible to substitute depolarization for stretch so as to produce the same overall PO time course. What we found, however, was that at a given voltage, stretch-accelerated current rise and decline almost identically—normalized current traces with and without stretch could be matched by a rescaling of time. Rate-limitation of the current falling phase by activation was ruled out. We hypothesize, therefore, that stretch-induced bilayer decompression facilitates an in-plane expansion of the protein in both activation and inactivation. Dynamic structural models of this class of channels will need to take into account the inherent mechanosensitivity of voltage-dependent gating.

Membrane Stretch Slows the Concerted Step prior to Opening in a Kv Channel

In the simplest model of channel mechanosensitivity, expanded states are favored by stretch. We showed previously that stretch accelerates voltage-dependent activation and slow inactivation in a Kv channel, but whether these transitions involve expansions is unknown. Thus, while voltage-gated channels are mechanosensitive, it is not clear whether the simplest model applies. For Kv pore opening steps, however, there is excellent evidence for concerted expansion motions. To ask how these motions respond to stretch, therefore, we have used a Kv1 mutant, Shaker ILT, in which the step immediately prior to opening is rate limiting for voltage-dependent current. Macroscopic currents were measured in oocyte patches before, during, and after stretch. Invariably, and directly counter to prediction for expansion-derived free energy, ILT current activation (which is limited by the concerted step prior to pore opening) slowed with stretch and the g(V) curve reversibly right shifted. In WTIR (wild type, inactivation removed), the g(V) (which reflects independent voltage sensor motions) is left shifted. Stretch-induced slowing of ILT activation was fully accounted for by a decreased basic forward rate, with no change of gating charge. We suggest that for the highly cooperative motions of ILT activation, stretch-induced disordering of the lipid channel interface may yield an entropy increase that dominates over any stretch facilitation of expanded states. Since tail current τ(V) reports on the opposite (closing) motions, ILT and WTIR τ(V)tail were determined, but the stretch responses were too complex to shed much light. Shaw is the Kv3 whose voltage sensor, introduced into Shaker, forms the chimera that ILT mimics. Since Shaw2 F335A activation was reportedly a first-order concerted transition, we thought its activation might, like ILTs, slow with stretch. However, Shaw2 F335A activation proved to be sigmoid shaped, so its rate-limiting transition was not a concerted pore-opening transition. Moreover, stretch, via an unidentified non–rate-limiting transition, augmented steady-state current in Shaw2 F335A. Since putative area expansion and compaction during ILT pore opening and closing were not the energetically consequential determinants of stretch modulation, models incorporating fine details of bilayer structural forces will probably be needed to explain how, for Kv channels, bilayer stretch slows some transitions while accelerating others.

FMRFamide and membrane stretch as activators of the Aplysia S-channel

The long-standing distinction between channels and transporters is becoming blurred, with one pump protein even able to convert reversibly to a channel in response to osmotic shock. In this light, it is plausible that stretch channels, membrane proteins whose physiological roles have been elusive, may be transporters exhibiting channel-like properties in response to mechanical stress. We recently described a case, however, where this seems an unlikely explanation. An Aplysia K channel whose physiological pedigree is well established (it is an excitability-modulating conductance mechanism) was found able to be activated by stretch. Here we establish more firmly the identity of this Aplysia conductance, the S-channel, as a stretch channel. We show that the permeation and fast kinetic properties of the stretch-activated channel and of the FMRFamide-activated S-channel are indistinguishable. We have also made progress in extending the kinetic analysis of the stretch channel to situations of multiple channel activity. This analysis implements a novel renewal theory approach and is therefore explained in some detail.

Stretch activation of the Aplysia S-channel

SummaryThe S-channel, a receptor-mediated K+ channel of Aplysia sensory neurons which functions in neuromodulation, bears a strong resemblance to the ubiquitous stretch-activated channels of snail neurons. Snail neuron stretch channels are stretch sensitive only in the patch, not at the macroscopic level, a situation which leaves open the question of their physiological role. If S-channels resemble snail stretch channels because both belong to the same general class of channels, the S-channel, too, should display stretch sensitivity in the patch. We show, using single-channel recording, that the S-channel can be activated by stretch. Furthermore, we show that Aplysia neurons in general have stretch-activated K+ channels. We suggest that the stretch-sensitive K+ channels of molluscan neurons and other preparations (e.g., Drosophila muscle, snail heart) are S-like channels, i.e., receptor-mediated channels which adventitiously exhibit mechanosensitivity in the patch.