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 plas ma membrane Ca2+ permeable cation channel were recorded using wide-field di gital imaging microscopy with fluo-3 as the Ca2+ indicator. These transient s were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp te chnique. This carton 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 locati ons on the cell membrane, with the duration of the rising phase matching th e duration of the channel opening. Moreover; these transients were only obs erved in the presence of sufficient extracellular Ca2+, suggesting that the y are due to Ca2+ influx from the bathing solution. The fluorescence transi ent is characterized by an initial fast rising phase when the channel opens , followed by a slower rising phase during prolonged openings. When the cha nnel closes there is an immediate fast falling phase followed by a slower f alling phase. Computer simulations of the underlying events were used to in terpret 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 nea r the channel when the channel opens or closes, while the slow phases are d ue to the diffusion of Ca2+ and Ca2+-bound fluo-3 into the cytoplasm. Trans ients due to short channel openings have a "Ca2+ spark-like" appearance, su ggesting that the rising and early falling components of sparks (due to ope nings of ryanodine receptors) reflect the fast phases of the fluorescence c hange, The results presented here suggest methods to determine the relation ship 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 plas ma membrane.