Visualization of Ca²⁺entry through single stretch-activated cation channels

Abstract

Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca 2؉ permeable when Ca 2؉ is the only charge carrier, it has not been clearly demonstrated that significant Ca 2؉ passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca 2؉ fluorescence transient (SCCaFT) arising from Ca 2؉ entry through a single SAC opening, we provide direct evidence that significant Ca 2؉ can indeed pass through SACs and increase the local [Ca 2؉ ]. Results were obtained under conditions where the only source of Ca 2؉ was the physiological salt solution in the patch pipette containing 2 mM Ca 2؉ . Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca 2؉ increase that occurred because of Ca 2؉ influx through voltage-gated Ca 2؉ channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence (''signal mass'') we determined that about 18% of the SAC current is carried by Ca 2؉ at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca 2؉ current at the resting potential. Such Ca 2؉ currents passing through SACs are sufficient to activate large-conductance Ca 2؉ -activated K ؉ channels and, as shown previously, to trigger Ca 2؉ release from intracellular stores.S tretch-activated channels (SACs) have been found in many cell types and are thought to be involved in various mechanical signal transduction mechanisms ranging from hearing, touching, and cell movement to smooth muscle contraction (for reviews, see refs. 1-3). SACs in smooth muscle have also been shown to be Ca 2ϩ permeable (4-6). This conclusion was obtained from patch-clamp ion substitution experiments where Ca 2ϩ was the only available carrier of the inward current. These studies also pointed out that the SAC conductance for Ca 2ϩ is much smaller than for monovalent cations. Therefore, it has not been clearly demonstrated whether significant amounts of Ca 2ϩ can indeed enter the cell through SACs when physiological salt solutions are used, or whether the fraction of the current carried by Ca 2ϩ is large enough to have a significant effect on the intracellular [Ca 2ϩ ]. Nevertheless, SACs have been considered essential for stretch-induced contractions and vascular smooth muscle myogenic responses (4, 6-8). Two roles for SACs were proposed: to pass cations (mainly Na ϩ ) leading to membrane depolarization, and to pass Ca 2ϩ to increase the cytosolic [Ca 2ϩ ] either directly or indirectly by triggering Ca 2ϩ release from stores.Indirect evidence that Ca 2ϩ could enter cells through SACs was obtained by using various Ca 2ϩ imaging techniques...

Imaging single-channel calcium microdomains

“…By employing high-speed, wide-field fluorescence microscopy Zou et al [17] were able to record localized fluorescence transients associated with the opening of sparsely-distributed stretch-activated cation channels in smooth muscle cells ( Fig. 2A).…”

“…Soon after, yet smaller 'fundamental' events (blips and quarks) were observed, and were interpreted to arise from the openings of single channels [12,14,15]. However, the first conclusive demonstrations of single-channel Ca 2+ signals were provided by Zou et al and Wang et al, [16,17] who combined optical and electrophysiological techniques to obtain simultaneous recording of current flow through a single open ion channel together with imaging of singlechannel Ca 2+ fluorescent transients (SCCaFTs).…”

“…Imaging techniques with improved resolution in both lateral (x−y) and axial (z) dimensions are thus expected to provide not only a better spatial localization, but also an enhanced temporal resolution by virtue of monitoring Ca 2+ -dependent fluorescence from tinier sampling volumes immediately adjacent to the channel. Several techniques have been used to image SCCaFTs, including wide-field microscopy [17,18], confocal linescan microscopy [16,19], and total internal reflection fluorescence microscopy (TIRFM) [20][21][22][23]; see [24][25][26][27] for reviews. Each methodology has particular advantages related to specific applications, but we concentrate on recent developments in TIRF microscopy as this is currently the most advantageous technique for imaging plasmalemmal Ca 2+ microdomains, permitting fast, twodimensional imaging of fluorescence signals within an exceedingly thin (ca.…”

Magnesium permeation through mechanosensitive channels: single-current measurements

“…Two roles are usually proposed: to pass cations (mainly Na + ) leading to membrane depolarization, and to pass Ca 2+ to increase the free cytosolic Ca 2+ concentration either directly or indirectly (by triggering Ca 2+ release from stores in muscle cells). Evidence for Ca 2+ permeability has been obtained using fluorescence measurements and single-current recordings at physiological Ca 2+ levels [15,34]. Measurements of Mg 2+ currents and permeability for MS channels imply that additional functional roles could be considered, particularly in cells of blood origin.…”

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

“…In addition, the signal mass of sparklets was determined. Details of the nP s and signal mass analyses can be found elsewhere (ZhuGe et al, 2000;Zou et al, 2002;Navedo et al, 2005Navedo et al, , 2006 …”