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

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 Ca2+ permeable when Ca2+ is the only charge carrier, it has not been clearly demonstrated that significant Ca2+ passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca2+ fluorescence transient (SCCaFT) arising from Ca2+ entry through a single SAC opening, we provide direct evidence that significant Ca2+ can indeed pass through SACs and increase the local [Ca2+]. Results were obtained under conditions where the only source of Ca2+ was the physiological salt solution in the patch pipette containing 2 mM Ca2+. 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 Ca2+ increase that occurred because of Ca2+ influx through voltage-gated Ca2+ 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 Ca2+ at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca2+ current at the resting potential. Such Ca2+ currents passing through SACs are sufficient to activate large-conductance Ca2+-activated K+ channels and, as shown previously, to trigger Ca2+ release from intracellular stores.