Dual stretch responses of mHCN2 pacemaker channels: accelerated activation, accelerated deactivation.

Mechanoelectric feedback in heart and smooth muscle is thought to depend on diverse channels that afford myocytes a mechanosensitive cation conductance. Voltage-gated channels (e.g., Kv1) are stretch sensitive, but the only voltage-gated channels that are cation permeant, the pacemaker or HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, have not been tested. To assess if HCN channels could contribute to a mechanosensitive cation conductance, we recorded I(HCN) in cell-attached oocyte patches before, during, and after stretch for a range of voltage protocols. I(mHCN2) has voltage-dependent and instantaneous components; only the former was stretch sensitive. Stretch reversibly accelerated hyperpolarization-induced I(mHCN2) activation (likewise for I(spHCN)) and depolarization-induced deactivation. HCN channels (like Kv1 channels) undergo mode-switch transitions that render their activation midpoints voltage history dependent. The result, as seen from sawtooth clamp, is a pronounced hysteresis. During sawtooth clamp, stretch increased current magnitudes and altered the hysteresis pattern consistent with stretch-accelerated activation and deactivation. I(mHCN2) responses to step protocols indicated that at least two transitions were mechanosensitive: an unspecified rate-limiting transition along the hyperpolarization-driven path, mode I(closed)-->mode II(open), and depolarization-induced deactivation (from mode I(open) and/or from mode II(open)). How might this affect cardiac rhythmicity? Since hysteresis patterns and "on" and "off" I(HCN) responses all changed with stretch, predictions are difficult. For an empirical overview, we therefore clamped patches to cyclic action potential waveforms. During the diastolic potential of sinoatrial node cell and Purkinje fiber waveforms, net stretch effects were frequency dependent. Stretch-inhibited (SI) I(mHCN2) dominated at low frequencies and stretch-augmented (SA) I(mHCN2) was progressively more important as frequency increased. HCN channels might therefore contribute to either SI or SA cation conductances that in turn contribute to stretch arrhythmias and other mechanoelectric feedback phenomena.

Studying the mechanosensitivity of voltage-gated channels using oocyte patches.

The mechanosensitivity of voltage-gated (VG) channels is of biophysical, physiological. and pathophysiological interest. Xenopus oocytes offer a critical advantage for investigating the electrophysiology of recombinant VG channels subjected to membrane stretch, namely, the ability to monitor macroscopic current from membrane patches. High-density channel expression in oocytes makes for macroscopic current in conventional-size, mechanically sturdy patches. With the patch configuration, precisely the same membrane that is voltage-clamped is the membrane subjected to on-off stretch stimuli. With patches, meaningful stretch dose responses are possible. Experimental design should facilitate within-patch comparisons wherever possible. The mechanoresponses of some VG channels depend critically on patch history. Methods for minimizing and coping with interference from endogenous voltage-dependent and stretch-activated endogenous channels are described.

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 tau(V) reports on the opposite (closing) motions, ILT and WTIR tau(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 ILT's, 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.

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.