Na channels and two types of Ca channels in rat pancreatic B cells identified with the reverse hemolytic plaque assay

Abstract

The reverse hemolytic plaque assay (RHPA) was used to study the secretory properties of single rat pancreatic B cells, and to identify insulin-secreting cells for patch-clamp experiments. In secretion studies using the RHPA, we find that the percentage of secreting B cells and the amount of insulin secreted per B cell increase as the glucose concentration is raised from 0 to 20 mM. Using the whole-cell variation of the patch-clamp technique, we find that identified B cells have three types of channels capable of carrying inward current: (a) tetrodotoxin-sensitive, voltage-dependent Na channels, which are nearly completely inactivated at -40 mV, (b) fast deactivating (FD) Ca channels, and (c) slowly deactivating (SD) Ca channels. We have shown that Na channels are functionally significant to the B cell, because tetrodotoxin partially inhibits glucose-induced insulin secretion. The properties of FD and SD Ca channels differ in several respects. FD channels deactivate at -80 mV, with a time constant of 129 microseconds, they are half-maximally activated near +15 mV, they do not inactivate during 100 ms, they conduct Ba2+ better than Ca2+, and they are very sensitive to washout during intracellular dialysis. SD channels, on the other hand, deactivate with a time constant of 2.8 ms, they are half-maximally activated near -5 mV, they inactivate rapidly, they conduct Ba2+ and Ca2+ equally well, and they are insensitive to washout.