Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 9

Ionic mechanisms responsible for action potentials in squid axon

Major Concepts and Objectives

1. Know the terminology describing voltage and current changes that occur during an action potential.

2. Understand the various components of the neuronal action potential wave form

3. Review the ionic basis of the action potential from squid axon

4. Understand ion channel gating and how Na and K channels contribute to the action potential

5. Know the molecular basis for channel gating that underlies the neuronal AP

6. Understand the effects temperature, [K+] and protease treatment on the AP waveform.

Readings

1. Rosenthal, J. J. and W. F. Gilly. Amino acid sequence of a putative sodium channel expressed in the giant axon of the squid Loligo opalescens. Proc Natl Acad Sci U S A 90: 10026-30, 1993.

2. Rosenthal, J. J. and W. F. Gilly. Identified ion channels in the squid nervous system. Neurosignals 12:126-141, 2003.

On the web: Nerve: http://nerve.bsd.uchicago.edu/ This web site provides a simulation of action potential generation and propagation in squid axon.

1 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 9

A. Action potentials and propagation 1. Definition of an action potential: An action potential (AP) is a rapid change in membrane voltage that produces a physiological response. Action potentials are all-or-none events, having waveforms that propagate with constant amplitude and time course independent of stimulus intensity. The response that follows an action potential depends on the cell type. In skeletal and cardiac muscle, action potentials trigger release of calcium leading to contraction. In nerve cells, action potentials trigger release of neurotransmitters that produce graded potentials in a post-synaptic nerve cell or end plate potentials in a skeletal muscle cell that is supplied by that nerve. 2. Propagation For electrical signaling to occur in excitable cells, action potentials must propagate along the cell membrane. This occurs by the spread of local currents from active (depolarized) regions of the cell to adjacent inactive (resting) regions of the cell. When the cell membrane becomes depolarized, positive charge entering the cell flows toward a more negatively charged area in an adjacent inactive (or resting) site. These local currents are responsible for bringing the adjacent region of the cell membrane to the threshold voltage, thus triggering another action potential. 3. Terminology:

 The cell membrane is said to depolarize when Vm becomes less negative than the referenced membrane potential.

 The cell membrane is said to repolarize when Vm moves toward the resting membrane potential.

 The cell membrane is said to hyperpolarize when Vm becomes more negative than the referenced membrane potential.

 Threshold potential: The membrane potential at which an action potential is initiated. The threshold potential is always less negative than the resting membrane potential. Figure 1: AP measurements  Inward current: By convention, an inward current is the flow of positive charge into the cell or negative charge out of the cell. Thus, inward currents depolarize the cell membrane.

 Outward current: By convention, an outward current is the flow of positive charge out of the cell or negative charge into the cell. Thus, outward currents repolarize or hyperpolarize the cell membrane. B. General anatomy of a neuronal action potential: The properties of the neuronal action potential (AP) are illustrated in Figure 1. This AP was elicited by current injection into a pyramidal neuron from the CA1 region of rat brain. The response to a sub-threshold depolarization is shown in

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red. Note the typically measured parameters of the AP waveform: threshold, defined as the most negative membrane potential that must be reached in order to elicit the all-or-none AP response; overshoot, difference between the peak voltage (Vpeak) and zero mV; spike height, difference between Vpeak and the most negative voltage attained during the after- hyperpolarization (AHP) and spike width, the width at half-maximal spike amplitude. As previously mentioned, action potentials are the result of coordinated opening, closing and inactivation of voltage gated Na and K channels that are located at the initial segment. Figure 2 shows the results of simulation of the action potential from squid axon which has served as an important model system for understanding the mechanisms that underlie the AP waveform.

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Figure 2: Anatomy of an action potential from the squid giant axon. Note that changes in membrane potential are shown in black, where as changes in Na and K conductances (gNa and gK) are shown in green and blue respectively (y-axis for each are also color coded). The equilibrium potentials for Na (green) and K (blue) are also indicated. The initial step (black trace) from -60 to -50 mV represents the threshold depolarization needed to activate the Na+ channels. Na channel activation (voltage-induced opening) produces the rapid increase in gNa that leads the voltage increase (upstroke). Na conductance then rapidly falls due to Na channel inactivation, a non-conducting conformational state of the channel. This fall in gNa combined with the slower increase in gK contributes to the repolarization phase of the AP and prevents the peak of the AP from reaching ENa. The K conductance reaches its peak at a later time when most of the Na channels are inactivated, is sustained for a longer period of time. This increase in K permeability above the normal resting K conductance produces an after-hyperpolarization response that drives the membrane potential close to the equilibrium potential for K+. As the voltage-activated K channels close (a process known as deactivation), the voltage returns to the original resting membrane potential. Some important consequences on the responsiveness of the neuron to a second stimulus occur as a result of gating properties and level of activity of Na and K channels that produce the AP:

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 Absolute refractory period (ARP): The period of time where a second action potential cannot be elicited no matter how intense the stimulus (indicated in red).  Relative refractory period (RRP): A time following the peak of an action potential where it is possible to elicit an action potential with a supra-maximal stimulus (indicated in orange).

Figure 3: Na and K channel gating events activation www.blackwellpublishing.com/matthews/channel.html deactivation C O C. Channel gating events during an AP r ecovery from inactivation The sequence of Na and K channel opening, inactivation I closing and inactivation events that result in an AP are shown above, moving from left-to-right (resting state → depolarization → repolarization → after-hyperpolarization → resting state). Note that the Na channel (GFLN1) has two gates, an activation gate (m-gate) and an inactivation gate (h-gate). At rest the m-gate is closed and the h-gate is open. Following threshold depolarization, the m-gate opens and Na+ enters the cell which increases depolarization towards ENa. With increasing depolarization, the h-gate closes and Na+ influx is inhibited as the Na channel enters the inactivated state. A gating scheme describing the conformational changes that take place for the Na channel is shown above. Starting from the closed state (C), threshold depolarization produces activation or opening (O) of the channel. Closing of the h-gate shortly afterwards results in inactivation (I). If the membrane voltage rapidly returns to the resting level prior to closing of the h-gate, then the channel will close (deactivation). Transition from the inactivated state to the closed state is called recovery from inactivation and this process takes place during the after- hyperpolarization phase of the AP.

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K channel gating involves transitions between closed and open states of the channel without inactivation. Many K channel subtypes however do exhibit inactivation and do contribute to the AHP response in mammalian neurons. For the squid axon, the slow rise in K+ conductance is due to voltage-dependent activation of Kv (SqKv1) channels, which requires opening of the n-gate. The subsequent decrease in conductance results from deactivation occurring when the n-gate closes. D. Ion channel subtypes involved in the squid axon AP Na channels: The molecular identity of the voltage-gated Na channel present in squid giant axon was determined by Rosenthal and Gilly (PNAS, 90:10026-30, 1993) using degenerate primers and PCR to probe a cDNA library from Loligo opalescens. A cDNA fragment was identified corresponding to a putative Na channel mRNA present in giant fiber lobes (GFLs) of the stellate ganglion (SG). The channel was named GFLN1 referring to its anatomical location within the GFLs. Sequence analysis revealed a high degree of identity with Nav- subunits from rat brain, electric eel electroplax and another squid Na channel SQSR cloned from the optic lobe of L. bleekeri. Overall similarity between the two squid channels was 32%, whereas similarity to eel electroplax was 43%. Greatest homology was associated with the membrane spanning domains, particularly S4, S5 and S6 along with the SS1 and SS2 segments linking S5 and S6. GFLN1 appears to be the only Na channel subtype present in squid giant axon and accounts for the voltage-dependent Na+ conductance. Figure 4: S4 and SS2 sequence homology K channels: Four related Kv1-type K channel (SqKv1) mRNAs have been detected in GFL and SG from L. opalescens. The structures are nearly identical except for the NH2-termini, but differ with respect to cellular localization with SqKv1A present in GFL, SqKv1B and D in SG, and SqKv1C undetectable within these structures. SqKv1A and B are the most prominent and exhibit delayed rectifier currents when expressed in Xenopus oocytes comparable to native delayed rectifier channels in the GFL. IK measurements obtained from oocytes are comparable to those recorded from GFL and SG

Figure 5: SqKv1 channels in GFL and SG Figure 6: SqKv1A and B currents

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neurons. Single channel properties for SqKv1A and SqKv1B are similar to the native squid K channels with a single channel conductance of 20 pS (after adjustment for differences in solution ionic composition), exhibit short latency and prominent bursting behavior, and inactivate over several hundred milliseconds.

+ E. Modulation of the AP waveform: Effects of temperature, [K ]o, pronase and tetrodotoxin (mS/cm2) gK+ gNa+ 100 20 40

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EK = -77 mV -100 -20 -40 0 1 2 3 4 5 6 7 8 9 10 Time (ms) Figure 7: Effects of increasing temperature from 6.3 to 25 oC on the squid action potential

Figure 7 shows the effects of temperature on the AP waveform using NERVE simulation software. The AP shown as a dashed line illustrates the duration of the AP at 6 oC (see Figure 2). When the temperature is increased to 25 oC, a dramatic change in the kinetics of the AP occurs. This is the result of temperature speeding up protein conformational transitions that open, close and inactivate the channels. This is directly reflected in the Na and K conductance changes shown in the green and blue traces respectively. Also note that increasing temperature O shifted both EK and ENa in opposite directions compared to conditions at 6 C (see figure 2). These changes are predictable since temperature is one of the terms in the Nernst equation.

Figure 8: Effects of extracellular [K+] from 10 mM to 35 mM on the AP. (mS/cm2) gK+ gNa+ 100 20 40

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+ with the dashed line is [K ]o = 10 mM. First note the effect on EK. This effect is consistent + + with a decrease in the [K ] gradient across the membrane. Clearly, increasing [K ]o reduced the amplitude of the AP. Also note that the increases in gNa and gK are also reduced. The decrease in gNa is due to depolarization of the resting membrane potential (RMP) produced by + increasing [K ]o, producing steady-state inactivation of a significant number of the Na channels. Thus fewer Na channels were available to generate depolarization after threshold is achieved. Consequently, less depolarization results in fewer K channels being activated after + the peak in gNa, but similar kinetics compared to the AP when [K ]o = 10 mM. Figure 9: Effects of pronase on the AP waveform (mS/cm2) gK+ gNa+ 100 20 40 gNa+ gK+ 75

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Figure 9 demonstrates the effects of M an enzyme (pronase) that was -75 E = -74 mV added to the intracellular fluid of -100 K -20 -40 squid axon for the purpose of 0 1 2 3 4 5 6 7 8 9 10 Time (ms) cleaving the inactivation gate away from the Na channel. Pronase (a mixture of serine proteinases secreted by Streptomyces griseus) treatment produces a significant change in the kinetics of the AP, specifically increasing its duration without altering the activation time course or the amplitude. The rate of repolarization however was reduced. This effect correlates well with the prolonged duration of gNa, which is what would be predicted if the h-gate is removed from the channel. Another effect is the increase in gNa amplitude, which makes sense since rapid inactivation no longer reduces Na+ influx prior to complete activation of the channel. Figure 10: Effects of tetrodotoxin (mS/cm2) gK+ gNa+ 100 20 40

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M blocker derived from a bacterium, -75 Vibrio alginolyticus, present within the E = -74 mV -100 K -20 -40 tissues of the pufferfish, various 0 1 2 3 4 5 6 7 8 9 10 species of blue-ringed octopus, certain Time (ms)

7 Scott M. O’Grady ANSC/PHSL 5700/PHSL 4700 Cell Physiology Lecture 9 toads and assorted invertebrates. Concentrations of the toxin in the nanomolar range can totally + abolish the AP by inhibiting Na channel activity. Interestingly, Nav isoforms in the heart appear to be less sensitive to tetrodotoxin than those present in nerve and skeletal muscle. Thus death often results from paralysis of the diaphragm, leading to respiratory insufficiency.

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