Imaging Ca²⁺ Entering the Cytoplasm through a Single Opening of a Plasma Membrane Cation Channel

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca²⁺ permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca²⁺ indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375–394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca²⁺, suggesting that they are due to Ca²⁺ influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca²⁺ and Ca²⁺-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca²⁺ and Ca²⁺-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a “Ca²⁺ spark-like” appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca²⁺ current, to study intracellular localized Ca²⁺ handling as might occur from single Ca²⁺ channel openings, and to localize Ca²⁺ permeable ion channels on the plasma membrane.