Lou Byerly

A Cytoskeletal Mechanism for Ca2+ Channel Metabolic Dependence and Inactivation by Intracellular Ca2+

Many different types of voltage-dependent Ca2+ channels inactivate when intracellular ATP declines or intracellular Ca2+ rises. An inside-out, patch-clamp technique was applied to the Ca2+ channels of Lymnaea neurons to determine the mechanism(s) underlying these two phenomena. Although no evidence was found for a phosphorylation mechanism, agents that act on the cytoskeleton were found to alter Ca2+ channel activity. The cytoskeletal disrupters colchicine and cytochalasin B were found to speed Ca2+ channel decline in ATP, whereas the cytoskeletal stabilizers taxol and phalloidin were found to prolong Ca2+ channel activity without ATP. In addition, cytoskeletal stabilizers reduced Ca(2+)-dependent channel inactivation, suggesting that both channel metabolic dependence and Ca(2+)-dependent inactivation result from a cytoskeletal interaction.

Calcium currents in internally perfused nerve cell bodies of Limnea stagnalis

1. When K+ is removed from both sides of the somal membrane of Limnea neurones, time‐dependent, voltage‐dependent outward currents are observed at positive potentials. These currents can be carried by Tris+ and tetraethylammonium (TEA+), as well as Cs+, but the Cs currents are several times larger. The Cs currents are not affected by external or internal TEA, but are strongly reduced by 4‐aminopyridine (4‐AP) and all Ca blockers tried.

The calcium channel

Abstract The application of intracellular perfusion and patch clamp techniques to the somal membranes of excitable cells has recently allowed rapid advancement in our understanding of the biophysical properties of the Ca 2+ channel. The activation of the Ca 2+ channel involves a series of slow and fast steps, while Ca 2+ current inactivation can result from a number of distinct processes. Recent detection of outward current through Ca + channels and instantaneous current measurements have provided information on the selectivity and current-voltage properties of open Ca 2+ channels. The ability of Ca 2+ channels to carry current appears to require as yet unidentified intracellular factors.

Characterization of proton currents in neurones of the snail, Lymnaea stagnalis.

1. Internal perfusion voltage‐clamp and inside‐out patch‐clamp techniques were used to study the voltage‐dependent H+ currents in snail neurone cell bodies. 2. In whole cells the voltage‐activated outward H+ current was measured 60 ms after stepping to +40 mV with an internal pH (pHi) of 5.9 and no internal K+([K+]i = 0), and the delayed K+ current was measured 60 ms after stepping to +40 mV with pHi = 7.3 and [K+]i = 74 mM. The mean H+ and K+ current densities were 14.6 +/‐ 7.8 and 38.2 +/‐ 14.0 nA/nF, respectively, giving a mean ratio of the H+ to K+ current of 0.4 +/‐ 0.2. There is not a strong correlation between the densities of the two kinds of outward currents found in different cells. 3. Inside‐out patch studies reveal that the H+ and K+ currents are distributed quite differently in the membrane. While 85% of all patches had K+ current, only five out of thirty‐eight patches studied had H+ currents. In those five patches the H+ currents measured at +30 mV ranged from 10.7 to 21.0 pA, and the ratio of the H+ and K+ currents at +30 mV was 0.83 +/‐ 0.38. The mean H+ and K+ currents for all thirty‐eight patches were 1.9 +/‐ 4.9 and 10.5 +/‐ 7.9 pA, respectively. 4. The current distribution patterns demonstrate that the H+ current does not flow through the delayed K+ current channels even though the two currents have similar voltage dependence and time course. 5. The relative ability of various extracellular divalent cations to block the H+ current was found to be Cu2+ approximately equal to Zn2+ greater than Ni2+ greater than Cd2+ greater than Co2+ greater than Mn2+ greater than Mg2+ = Ca2+ = Ba2+. Since 100 microM‐Zn2+ blocks the H+ current more than it blocks the Ca2+ current, it can be used to reduce the contamination of Ca2+ current measurements by the H+ current. 6. The magnitude of the H+ current has a stronger temperature sensitivity than does the magnitude of the delayed K+ current. The Q10 of the H+ current magnitude is 2.1 +/‐ 0.4, while the Q10 of the K+ current magnitude is 1.4 +/‐ 0.04. This suggests a higher activation energy may be involved in the conduction of the H+ current than for K+ current. 7. The smooth time course of the H+ current measured in patches indicates that the size of the unitary H+ current is very small.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ channel Ca2+-dependent inactivation in a mammalian central neuron involves the cytoskeleton

Ca2+ channel inactivation was investigated in acutely isolated hippocampal pyramidal neurons from adult rats and found to have a component dependent on intracellular Ca2+. Ca2+-dependent inactivation was identified as the additional inactivation of channel current observed when Ca2+ replaced Ba2+ as the current carrying ion, and was found to be an independent process from that of Ba2+ current inactivation based on three lines of evidence: (1) no correlation between Ca2+-dependent inactivation and Ba2+ current inactivation was found, (2) only Ca2+-dependent inactivation was reduced by intracellular application of Ca2+ chelators, and (3) only Ca2+-dependent inactivation was sensitive to compounds which alter the cytoskeleton. Drugs which stabilize (taxol and phalloidin) and destabilize (colchicine and cytochalasin B) the cytoskeleton altered the development and recovery from Ca2+-dependent inactivation, indicating that the neuronal cytoskeleton may mediate Ca2+ channel sensitivity to intracellular Ca2+. Ca2+-dependent inactivation was not associated with a particular subset of Ca2+ channels, suggesting that all Ca2+ channels in these neurons are inactivated by intracellular Ca2+.

Spider toxins selectively block calcium currents in drosophila

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.

Calcium Channel Diversity

In recent years a great deal of evidence has accumulated that demonstrates the large amount of diversity that exists between Ca channels. By Ca channels we mean membrane pores that are opened by depolarization, allow Ca2+ to flow down its electrochemical gradient when the channel is open and show a standard selectivity between different divalent cations. Ca2+, Ba2+ and Sr2+ can pass through Ca channels, while Co2+ and Mg2+ cannot. Cd2+, Co2+, Ni2+ and Mn2+ block the channel, but some of the blockers, e.g., Mn2+, can themselves carry current through the channel. This definition of Ca channels excludes several interesting channels which have been shown to allow Ca2+ to enter cells.

Voltage-independent barium-permeable channel activated in Lymnaea neurons by internal perfusion or patch excision.

SummaryIsolated nerve cells fromLymnaea stagnalis were studied using the internal-perfusion and patch-clamp techniques. Patch excision frequently activated a voltage-independent Ba2+-permeable channel with a slope conductance of 27 pS at negative potentials (50mm Ba2+). This channel is not seen in patches on healthy cells and, unlike the voltage-dependent Ca channel, is not labile in isolated patches. The activity of the channel in inside-out patches is unaffected by intracellular ATP, Ca2+ below 1mm or the catalytic subunit of cAMP-dependent protein kinase but is reversibly blocked by millimolar intracellular Ca2+ or Ba2+. The channel can be activated in on-cell patches by either internal perfusion with high Ca2+ or the long-term internal perfusion of low Ca2+ solutions not containing ATP. These channels may carry the inward Ca2+ current which causes a regenerative increase in intracellular Ca+ when snail neurons are perfused with high Ca2+ solutions. High internal Ca2+, or long periods of internal perfusion with ATP-free solutions, induces an increase in a resting (−50 mV) whole-cell Ba2+ conductance. This conductance can be turned off by returning the intracellular perfusate to a low Ca2+ solution containing ATP and Mg2+. The activity of this channel appears to have an opposite dependence on intracellular conditions to that of the voltage-dependent Ca channel.

Measurement of Calcium Channel Inactivation Is Dependent upon the Test Pulse Potential

We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies.