The purpose of this study was to examine the time course and kinetics of L-type Ca2+ current (ICa,L) from pituitary GH3 cells in response to various action potential (AP) waveforms using the whole-cell configuration of the patch-clamp technique. The major findings in this study are: 1. ICa,L evoked during the AP waveform exhibited an early and a late component. The early component of ICa,L occurred on the rising phase of the AP, whereas the late component coincided with the falling phase. 2. A prolongation of the falling phase of APs led to an increase in Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced. 3. When the peak voltage of AP waveforms was prolonged without changing the rising and falling phases, the amplitude of the late components was significantly increased. 4. ICa,L was also found to inactivate during a train of AP waveforms. When Ba2+ ions were used as the charge carriers, current inactivation during a train of APs was decreased. 5. The amplitude of ICa,L evoked by the AP templates with irregular bursting pattern was inactivated. 6. When spontaneous APs with the depolarizing potentials were replayed to GH3 cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory. 7. When cells were exposed to thyrotropin releasing hormone (TRH; 10

Diversity in the expression of ion channel proteins among neurons allows a wide range of excitability, growth and functional regulation. Ether-a-go-go (EAG), a member of the voltage-gated K+ channels, was characterized by spontaneous firing in nerve terminals and enhanced neurotransmitter release. In situ whole-cell patch-clamp recordings performed from the somata of Drosophila larval thoracic aCC motoneurons revealed spontaneous spike-like events in eag mutants. Spontaneous events were absent in wild type motoneurons. Spikes evoked by somatic current injection in to the cell body were not altered and comparable to wild type. Spontaneous spike-like events could be due to increased synaptic drive or altered intrinsic excitability of the motoneuron. Reduction of EAG function with selective expression of eag double stranded RNAi transgene in motoneurons only did not cause spontaneous spike-like events or alter evoked firing. This suggests increased synaptic drive contributes to spontaneous events.Both transient and sustained voltage-activated K+ currents, each with Ca++-sensitive (IA(Ca) and IK(Ca)) and Ca++ -insensitive components (IA and IK), were isolated in thoracic aCC motoneurons. In wild type motoneurons, IA was larger than IA(Ca). Conversely, IK(Ca) was larger than IK. Both eag mutants and eag RNAi expression resulted in a decrease in IA , IK and a slow sustained K+ current. Further, EAG and Shal demonstrate a potential functional interaction and contribute to IA. The voltage sensitivity for inactivation was reduced in Shal only and EAG-Shal double knock down compared to controls and EAG only knock down. In addition, a Ca++ sensitive EAG dependent K+ current was blocked by cAMP. Thus, both voltage-dependent and modulatory functions of EAG influence excitability in motoneurons.Firing properties and K+ currents distinguish aCC motoneurons in thoracic segments, T1 and T3. T3aCC had a shorter delay to spike, higher input resistance and were more easily recruited than T1aCC. T1aCC had a larger IA than T3aCC, but comparable IA(Ca). IK(Ca) was larger in T3aCC compared to T1aCC. These differences reflect cell-specific ion channel distribution that could contribute to patterned segmental motor output.

Zugl.: Giessen, Universiẗat, Diss., 2007.

Zugl.: Freiburg (Breisgau), Univ., Diss., 2009

Thesis (Ph. D.)--University of Chicago, Dept. of Pharmacological and Physiological Sciences, August 1999. / Includes bibliographical references. Also available on the Internet.

Mainz, Univ., Diss., 2001.

Abstract In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and functional adaptations. Thus far, many adaptations for night vision in the compound eyes of nocturnal insects have been described, but little is known about the mechanisms underlying the electrochemical signalling in their photoreceptors. In this thesis, whole-cell patch-clamp and mathematical modelling are utilised to study basic electrical properties and ionic currents in photoreceptors of two nocturnal insects, the American cockroach Periplaneta americana and the field cricket Gryllus bimaculatus. Photoreceptors in both species showed large input resistance, membrane capacitance and phototransduction gain (large single photon responses) compared with most studied diurnal insects, providing improved sensitivity to light. The photoreceptors also expressed two voltage-sensitive outward currents: a transient current and a sustained current. The cricket photoreceptor expressed a dominating transient current, which is a typical characteristic for insects adapted for slow vision in dim light. By contrast, in the majority of cockroach photoreceptors the sustained current dominated, which is more common among fast diurnal species. Model simulations indicated that the sustained current is necessary for improved photoreceptor dynamics. Examination of light-induced currents suggested that the functional variability in cockroach photoreceptors is in part derived from variations in the total area of the photosensitive membrane. Recordings of light-induced currents also revealed that the cockroach light-gated channels are only moderately Ca2+-selective and that the polarisation-sensitive photoreceptors of the cricket may utilise phototransduction machinery in some details different from that in regular photoreceptors. Furthermore, the dynamics and information transfer rates of polarisation-sensitive photoreceptors in the cricket were clearly inferior to their regular counterparts, suggesting that they are not necessary for image formation.

At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at -70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (K(Ca)) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K(Ca) activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V(m)) and 145 ± 13 pS at negative V(m). Glibenclamide (50 μM), Ba2+ (1 mM) and quinine (100 μM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 μM. The identity of this K+ channel with an ATP-sensitive K+ channel (K(ATP)) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 μM). We conclude that plasma membranes of HTB-9 cells contain the K(Ca) and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked K(ATP).

No description available.