Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Gap junction (GJ) channels are dynamic intercellular structures that open and close (gate) in response to cellular signals (e.g. voltage), and may be important in regulating cell-to-cell communication in insect development. This thesis presents one of the first biophysical characterizations of GJ channels in insect cell pairs. Double whole-cell voltage-clamp techniques were employed on epidermal cells from the larval flour beetle, Tenebrio molitor, to measure the conductances and substate gating properties of individual GJ channels and the effects of voltage and ATP on gating. In addition, these techniques were used to examine the early events of rapid GJ formation between hemocyte couplets from the cockroach, Periplaneta americana. For this, methods were developed for isolating epidermal cells and for preventing rapid flattening of hemocytes by using lipophorin-coated coverlips.;GJ formation between hemocyte couplets occurred within one second after contact and junctional conductance (G{dollar}\rm\sb{lcub}J{rcub}{dollar}) increased in steps of {dollar}\sim{dollar}345 pS. Each step corresponds to a new channel added to the growing gap junction.;Pharate pupal epidermal (PPE) and newly-moulted epidermal (NME) cells suitable for whole-cell recording were isolated. Single cells from both stages had large nonjunctional resistances ({dollar}>{dollar}5 G{dollar}\Omega{dollar}). NME but not PPE cells expressed a hyperpolarization-activated inward current.;Isolated PPE and NME cell pairs had functional gap junctions. G{dollar}\rm\sb{lcub}J{rcub}{dollar} dropped during whole-cell recording, however, adding 5 mM ATP but not nonhydrolysable ATP analogues to the pipette solution slowed uncoupling suggesting that ATP probably acts through an ATP-utilizing enzymatic process.;G{dollar}\rm\sb{lcub}J{rcub}{dollar} in epidermal cells is mostly membrane-potential (V{dollar}\rm\sb{lcub}M{rcub}{dollar}) dependent, although transjunctional-voltage dependence is also present.;Conductances of individual GJ channels in poorly-coupled PPE cell pairs were large (197 to 403 pS). This GJ channel is one of the first described capable of existing in a range of partially-closed substates.;GJ-channel gating mechanisms presented herein may be important in regulating cell-to-cell communication in insects. For instance, V{dollar}\rm\sb{lcub}M{rcub}{dollar}-dependence may provide a mechanism for controlling the overall level of coupling. Moreover, GJ-channel substates may account for selective regulation of junctional permeability in developing tissues.



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