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

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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.